Increases Risk of Colorectal Cancer NAT1

Polyadenylation Polymorphism in the Acetyltransferase 1 Gene ( NAT1) Increases Risk of Colorectal Cancer Douglas A. Bell, Elizabeth A. Stephens, Trisha Castranio, et al. Cancer Res 1995;55:3537-3542. Published online August 1, 1995.

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[CANCER RESEARCH 55, 3537-3542,

August 15, 1 G) at nt 1098 that created a partial Mboll restriction site. Subsequent PCR

1323F 860

TTTTTACTAT

TTAGAATAAG

GAGTAAAACA

910

Mboll AGCTCACCAG TTATCAACTG ACGACCTATC ATGTATCÃ•TCTGTACCCTTA

960

Mboll CCTTATTTTG AAGÃ„AAATCCTAGACATCAA ATCATTTCAC CTATAAAAAT

1010

GTCATCATAT ATAATTAAAC AGCTTTTTAA AGAAACATAA CCACAAACCT || a

1060

TTTCAAATAA

TAATAATAAT

AATAATAATA

ATCTTGTCTA

TTTGTCATCC

a Mboll1

Fig. 2. Ge! Electrophoresis of NATI genotypes. M (left and right lanes ), marker (Ã•C174Him/Ill; United States Biochemical, Cleveland, OH); Lane l, NAT1*4/NAT1*4; Lane 2, NATI*4/NATi*IO; Lane 3, NAT1*3/NATI*4; Lane 4, NATI*10/NAT1*IO; Lane 5, NAT1*4/NATI*11; Lane 6, NATI "IO/NAT 1*11; Lane 7, NATI"! I/NATI"! 1; B, blank (no DNA negative control), a, after Mboll digestion and electrophoresis on a high resolution agarose gel (4% Metaphor; FMC Bioproducts, Rockland ME), homozygous genotypes for the NAT1*4, NATI"IO, and NAT1*I1 alÃ-eles produced distinct band patterns (Lanes 1, 4, and 7, respectively). Mboll digestion of the NATI "4 alÃ-elecut the PCR product into fragments of 105, 71, 45, and 26 bp. Digest of a NATIfIO alÃ-eleresults in fragments of 131, 71, and 45 bp. The NATI*II alÃ-elecan be distinguished by observation of a 9-bp mobility shift of the 131-bp band to 122 bp (Lanes 5,6, and 7). Lane 5, presence of a band at 131 bp due to poor Mboll digestion of the NATI*4/NAT1*10 heteroduplex PCR product. HÃ©tÃ©rozygotes produce the predicted composite patterns. Note that the RFLP method fails to distinguish between the NATI*IO and NATI*} alÃ-eles,b, AS-PCR discriminates between an "A" at nt 1088 (NAT1*10) and a "T at nt 1088 (NATI*3 and NATI*4). The 268-bp band is 0-globin. In the upper row of bands, the 223-bp band indicates the presence of the NAT1*4 alÃ-ele.In the lower row of bands, the 223-bp band indicates the presence of the NAT! "10 alÃ-ele.This approach cannot easily distinguish individuals with the NATl*ll alÃ-ele(9-bp deletion).

AATGÃ•CTTTT AAAGATGGCC V\ak;V2t _9L

amplification of the NAT1*4 alÃ-elewith this primer resulted in a PCR product 1110

TGTGGTTATC TTGGAAATTG GTGATTTATG CTAGAAAGCT TTTAATGTTG ÃŽ536R

Fig. I. NATI gene sequence in the 3'-untranslated region near the polyadenylation signal. The alÃ-eles we detected were: NAT1*4, NATI*3, NAT1*10, and NAT1*I1. NATI* 10. the "at risk" rapid alÃ-ele,has nt 1088 = "A" and nt 1095 = "A" (the change at nt 1088 alters the mRNA polyadenylation signal, and the change at nt 1095 alters a partial Mholl restriction site). For the NAT1*11 alÃ-ele,it is not possible to determine exactly which 9-bp are deleted. See text for additional information; alÃ-elenomenclature is explained in Ref. 23. Variant nucleotides are in lowercase above the sequence. Restriction enzyme sites are bold and italicized. PCR primer locations are marked by lines (sense primers are above the sequence, antisense are below the sequence), t, partial Mboll restriction site; a full site is formed when a NAT!*4 alÃ-eleis amplified by PCR primer N1536R containing a Tâ€”> G substitution at nt 1098.

containing a new MeoII restriction site (Fig. 1). PCR conditions for the NATI PCR-RFLP method were the same as for NAT2, except for the following: AMTV-specific primers N1306F (5'-cta ttt aga ata agg agt aa) and N1536R (5'-aca ggc cat ctt tag aa), 2.0 mM MgCl, in a volume of 30 /j.1, and the PCR was denatured at 94Â°Cfor 4 min and subjected to 30-35 cycles of 94Â°Cfor 30 s, 42Â°Cfor 30 s, and 72Â°Cfor 45 s. A final 72Â°Cextension for 5 min was performed. Recently, we have also used a new forward primer N1208F (5'-gac tct gag tga ggt aga aat a; annealing at 52Â°C)with increased specificity for NATI. The PCR fragment patterns that are produced by each genotype are displayed in Fig. 2a. To verify genotype assignments, we carried out direct sequencing of the PCR products with the Applied Biosystems Prism DyeDeoxy terminator sequenc-

3538


NATI POLYMORPHISM

AND COLORECTAL

ing kit and the ABI 373A DNA sequencer, following the manufacturer's protocol (Applied Biosystems, Inc., Foster City, CA). We sequenced the NATI 3'-untrans-

CANCER

Table 1 AlÃ-elefrequencies

lated region for 40 individuals in this study who had a variety of genotypes (as well as ~80 individuals from other projects). We discovered that the NATI*3 alÃ-ele exists ("A" at nt 1095 but "T" at nt 1088) and that -3% of subjects carry this alÃ-ele.The PCR-RFLP method misclassifies these individuals as NATI* 10. We then developed an AS-PCR method to confirm the presence oÃ-NATI* IO alÃ-eles. This method used parallel PCRs for each DNA sample, with 5' primer nl323 (5'-taa aac aat ctt gtc tat ttg) in combination with either primer VÂ¡aK (5'-gcc ate ttt aaa a g/t a cat tta) or primer V2t (5' gcc ate tu aaa a t a cat ttt) (20 pmol each). Conditions were the same as above, except that 2.5 mM MgCl2 was used in a volume of 30 /xl, and the PCR was denatured at 94Â°Cfor 4 min and subjected to 30 cycles of 94Â°Cfor 30 s, 52Â°Cfor 30 s, and 72Â°Cfor 45 s. A final 72Â°C extension for 5 min was performed. Primers for ÃŸ-globin(5 pmol) were added to this PCR to produce a 268-bp reference band in all AS-PCR reactions (as in Ref. 24). The PCR product from the NATI AS-PCR was 223 bp. Positive and negative controls were included in each set of PCR analyses. Results for this technique with the various genotypes are shown in Fig. 2b. The a priori hypothesis was that the NAT1*10 alÃ-elewas the "at-risk" alÃ-ele.Our NATI genotype/phenotype compar isons suggest that the NAT1*10 alÃ-elecould be considered a "rapid" alÃ-elerelative to the other alÃ-eles.3In the data tables, NAT1*3, NAT1*4, and NATI*]] genotypes are arbitrarily labeled "slow."

RESULTS Colorectal case and control groups had similar frequency distribu tions for age and sex (cases: mean age, 63 years; male, 52%; female, 48%; controls: mean age, 63 years; male, 52%; female, 48%). Among colorectal patients, 33% were smokers at the time of diagnosis, but smoking status as an independent risk factor could not be assessed because this information was not available for the control population. However, among cases, smoking frequency was not associated with differences in tumor stage or grade. Data for other colorectal cancer risk factors was not available; therefore, adjustment for these factors was not conducted. Table 1 displays calculated NATI alÃ-elefrequencies for the groups in this study. Variant NATI alÃ-ele frequencies (NAT1*10 and NATI*il) in this English control group were similar to those ob served in a European-American population sample from North Caro lina.4 The alÃ-elefrequency distribution among colorectal cancer pa tients differed from the control group. The NAT1*4 and NAT1*11 alÃ-eleswere less frequent among cases, whereas the NATI* 10 alÃ-ele was more frequent among cases. Pooling the rare NAT1*3 alÃ-eleswith NAT1*4 alÃ-eles,the overall alÃ-elefrequency distributions among cases and controls were significantly different (P = 0.03, 2 degrees of

RISK

among the colorectal cancer and control populations for the 4 NATI alÃ-eles

NATI"4" (n)

NAT1*3

NATl'll

Total chromosomes

(n)

(n)

tested

Colorectal cancer 0.72(291) patients

0.02(8)

0.24(98)

0.017(7)

404

Staffordshire

0.03(6)'

0.16(36)

0.045(8)

224

0.77(174)*

(n)

NATI*IO

control patients " For nomenclature, see (Ref. 23); NATI*4, NAT1*3, and NAT1*11 are presumably "slow" alÃ-eles;NATI "IO is putatively a "rapid" alÃ-ele. * Four alÃ-elecomparison, x2 = 7.33; P = 0.06; 3 df. ' Three alÃ-elecomparison (NATI"4 and NATl*3 pooled), *2 = 7.0; P = 0.03; 2 df.

NAT1*10 alÃ-ele)and relative to NATI*!I 3.8; P = 0.023).

containing genotypes (OR,

Tables 3 and 4 display the NATI and NAT2 genotype frequencies among the hospital-based controls and colorectal cancer cases. The frequency of genotypes containing the NAT1*10 rapid alÃ-eleincreased from 29% among controls to 44% among cases (Table 3). Overall, individuals with an altered NATI polyadenylation signal (NAT1*10 alÃ-ele)had a 1.9-fold increased risk for colorectal cancer (95% CI, 1.2-3.1; P = 0.009). Following stratification of cases by tumor stage (Duke's staging classification), the frequency of at risk NATI geno types was increased among those with higher stage tumors relative to the control patients or to colorectal patients with lower stage tumors (Table 3). The frequency of NAT!*10 genotypes was significantly higher among stage B (OR, 1.9; 95% CI, 1.1-3.4; P = 0.03) and stage C cases (OR, 2.5; 95% CI, 1.3-4.7; P = 0.006) but not among stage A cases when compared against the control population. This trend toward higher stage tumors among individuals with the NAT1*10 genotypes was significant (Armitage-Doli trend test, P = 0.0015). Similarly, NATI *10 genotypes were more frequent among those with tumors that were graded as moderately or poorly differentiated (data not shown). For NAT2 genotypes (Table 3), individuals with 2 nonfunctional alÃ-eleswere classified as slow acetylators, whereas individuals with 1 or 2 functional alÃ-eleswere classified as rapid acetylators (see Refs. 7-11). NAT2 rapid acetylator genotypes were slightly higher among cases compared to control subjects (OR = 1.1), but this difference was not significant. There was also a trend toward a higher frequency of NAT2 rapid acetylator genotypes among higher stage tumors, but this increase did not reach significance (P = 0.26). Slow acetylators were overrepresented among individuals with Duke's A stage tumors,

freedom). Table 2 displays the frequencies of NATI genotypes among colo rectal cancer cases and controls. The NATI *10 alÃ-ele,containing the

but there were very few cases in this group. The frequencies of NATI rapid genotypes relative to tumor location were: ascending, 37% (22 of 59); transverse, 50% (4 of 8); descend altered polyadenylation signal, was considered to be the a priori at risk alÃ-ele.3Genotypes containing the NAT1*10 alÃ-ele(hÃ©tÃ©rozygotes ing, 51% (28 of 55); and rectal, 45% (29 of 65). NATI rapid genotypes were less frequent among those with a tumor in the ascending colon and homozygotes) were more frequent among colorectal cancer cases (37%); however, the lower frequency was not significantly different (40 and 4%, respectively) compared to the control group (26 and relative to other locations (P = 0.15). No significant differences in 2.7%, respectively). There was a 1.8-fold risk associated with inher NAT2 genotype frequency were observed with regard to location of iting either of these genotypes. Genotypes including the NATI*!] tumor. Among the 178 cases for whom we had smoking data (yes or alÃ-elewere less frequent among cases (2.5%) relative to controls (6%), but this difference was not significant (OR, 0.6; P = 0.26). Table 2 no response), we tested for differences in genotype frequency between smokers and nonsmokers (case-only analysis, see Ref. 22). There was also shows the risk calculation for the presumptive rapid genotypes a higher frequency of NAT1*10 genotypes among cases who were relative to all presumptive slow genotypes (OR, 1.9; P = 0.009; any smokers (52%, 29 of 56) relative to nonsmoking cases (41%, 50 of 122), but this difference was not significant (OR, 1.5; 95% CI, 3 Data from our laboratory indicate that the NATI*IO alÃ-ele,containing the altered 0.8-2.9; P = 0.19). Similarly, NAT2 rapid acetylator genotypes were

polyadenylation signal, is associated with higher tissue levels of NATI enzyme activity relative to the NAT1*4 alÃ-ele(D. A. Bell, A. Hirvonen, A. Badawi, N. P. Lang, K. Ilett, and F. F. Kadlubar. Polymorphism in the NATI polyadenylation signal: association of the NATI*IO alÃ-elewith higher W-acetylation activity in bladder and colon tissue samples. Submitted for publication.). No data were available for the NATI "II alÃ-ele. 4 D. A. Bell, unpublished data.

slightly increased among smoking cases (52%) relative to nonsmok ing cases (45%; OR, 1.3; 95% CI, 0.7-2.5; P = 0.4).

Table 5 shows the risk associated with combinations of NAT1/NAT2 genotypes when the NATI s\ow/NAT2 slow genotypes 3539


NATI POLYMORPHISM AND COLORECTAL CANCER RISK Table 2 NATI genotvpe frequencies

among colorectal cancer cases and controls

RapidNAT1*WINAT1*W

homozygoteColorectal 202)Control cancer (n =

(8)2.7%

112)Risk (n =

(3)1.8Â° (0.5-6.9)Iff1

CI)Risk (OR, 95%

hÃ©tÃ©rozygote homozygote2.5% or homozygote

(82)26.8%

(5)6.3%

(30)1.8*

(7)0.6r

(1.1-3.1).2-3.1).2-12.1)Slow

NATI *4(or NATI *3) (107)64.3% (72)1

(0.24-1. 47)53.0%

(I3.8'

CI)Risk for any NAT1*10 alÃ-elevs slow genotypes (OR, 95%

(1genotypesNATI

for any NATI*IO alÃ-elevs any NATI*I14.0% "P bP cP dP ' P

genotypesNAT1*1I "10 hÃ©tÃ©rozygote40.6%

= 0.39, risk relative to NAT1*4/NAT1*4 (or NAT1*3). = 0.016, a NAT1*IO/NATI*1I hÃ©tÃ©rozygote was included in both cases and controls. = 0.26. - 0.009. This compares risk of colorectal cancer for individuals who have one or two NAT1*10 alÃ-elesv.v slow genotypes (NATI*! 1 alÃ-eleswere pooled with slow alÃ-eles). = 0.023. This compares risk of colorectal cancer for individuals who had one or two NAT1*IO alÃ-elesvs those with NATI*I1 genotypes.

were considered the reference group. There was no apparent increased risk for the individuals who were NATI inpid/NAT2 slow (OR = 1.3) or NAT2 rupid/NATl slow (OR = 0.7). Among those with NAT! rapid/'NAT2 rapid genotypes, there was a significantly increased risk of 2.0 (95% CI, 1.1-3.8). Similarly, among those with NAT2 rapid acetylator genotype, NAT! rapid genotypes had a 2.8-fold increased risk relative to NATI slow acetylators (95% CI, 1.4-5.7; P = 0.003). This suggests a possible gene-gene interaction between NATI and NAT2. Using logistic regression to model this interaction (NATI x NAT2), we found a 2.2-fold elevated risk associated with the inter action term; however, this risk did not reach significance (OR, 2.2; 95% CI, 0.8-6.1; P = 0.12). DISCUSSION In this first examination of the relationship between NATI poly morphism and cancer, we found that inheritance of a variant NATI polyadenylation signal (NAT]*IO alÃ-ele)conferred a 1.9-fold in creased risk for colorectal cancer among all patients in this study (P = 0.009). Colorectal cancer patients with the NAT1*W alÃ-elewere more likely to have an advanced stage tumor (Duke's B or C). In addition, there appears to be a combined effect for NATI rapid genotypes and NAT2 rapid genotypes. Because we have no information about

Table 4 Comparison of NAT2 genotype frequency in colorectal cancer cases and controls T2Rapid48% 95%confidence ratio interval;P value)OR Colorectalcancer(n

(96)45%

(106)55%

(50)28%

(62)72%

=(0.71-1.8;P 1.1 0.62)OR =

202)Control(n =

112)Analysis = bytumor stage(n 183)Duke's = ADuke's

= (0.16-1.4;P 0.48 0.19)OR = = 0.96 (0.56-1.7;P (42)54%* (54)46% 0.9)OR= = (38)GenotypeSlow"52% CNA 1.47(0.81-2.7;P (32)Risk(odds = 0.21) "AM72 slow acetylator genotypes are Ml/Ml, M1/M2, M1/M3, M1/M4, M2/M2, (5)44%

(13)56%

BDuke's

M2/M3, M2/M4, M3/M3, M3/M4, M4/M4 (11, 23). '' Trend analysis was carried out on the frequency of NAT2 rapid genotypes among the 3 stage categories and including controls (4 groups), P = 0.27; or just within case groups (3 groups), P = 0.036.

Table 5 Analysis of gene-gene interaction Table 3 Risk associated with A'AT'I"10 genotypes among colorectal cancer cases and slow CI)0.7* OR (95%

controls with analysis by tumor stage Risk

NATI Genotype Rapid" (NAT1*W)

Slow

Colorectal cancer (n = 202)

44% (90)

56% (112)

Control (n= 112)

29% (33)

71% (79)

Analysis by tumor stage (n = 183) Duke's A

(odds ratio, 95% confidence interval; P value) OR=

1.92(1.2-3.1; P = 0.009)

78% (14)

3 stage categories and included controls (4 groups), P = 0.0015; or just cases (3 P = 0.052.

NAT2 slow (0.4-1 .3)NATI NAT2 rapid Risk of NATI "10 among M472 rapid NATI X NAT2 gene-gene interactionNATI Â°P = 0.25. * P = 0.49. CP = 0.031. d P = 0.003. e P = 0.12. This analysis tested the significance of the NATI effect among NAT2 rapids relative to the NATI effect among AM72 slows, (i.e., tests if 1.3 is significantly different from 2.8), P = 0.12.

OR = 0.7 (0.2-2.3; P = 0.58) Duke's B OR= 1.9(1.1-3.4; 43% (41) 57% (54) P = 0.03) Duke's C 50%* (35) 50% (35) OR = 2.5 (1.3-4.7; P = 0.005) " Genotypes containing NAT1*4, NATI*}, and NATI"! I alÃ-eleswere pooled as slow genotypes. Genotypes containing the NATI "Â¡OalÃ-ele(hÃ©tÃ©rozygote or homozygote) were considered rapid. b Trend analysis was carried out on the frequency of NATI rapid genotypes among the 22% (4)

rapid OR CI)1.3" (95% (0.6-2.7) 2.01' (1.1-3.8) 2.8''(1.4-5.7) 2.2'' (0.8-6.1)

possible dietary or environmental intake of carcinogens (or other risk factors that could potentially confound these findings), this study cannot directly address the specific mechanism of risk for NATI in colon carcinogenesis. Indeed, there is still uncertainty regarding the strength of the associations between various dietary, or other, exposures and colo rectal cancer (1, 2, 18-21). Although this limits our conclusions regard

ing the causal relation between NATI and colorectal cancer, we can offer a number of reasonable explanations. Recent studies indicate that the NATI enzyme is expressed groups), in colonie mucosa (25), indicating the potential for involvement in 3540


NATI POLYMORPHISM

AND COLORECTAL

carcinogen metabolism in the colon. Our preliminary results suggest that NAT 1*10 may lead to higher tissue levels of NATI enzyme,3

CANCER

RISK

genes is far from clear. Some proposed pathways for activation of arylamines and heterocyclic amines involve both NATI and NAT2 enzymes (6, 14, 30), and the effect we observe with combined NATI and NAT2 rapid genotypes may be a hint that these pathways may be important in colorectal carcinogenesis. Because of the epidemiologi cal limitations of this study, the finding that a NATI alÃ-eleconveys significant risk in colon cancer must be considered preliminary. How ever, the result suggests several new hypotheses regarding the NATI gene, its role in carcinogen metabolism, and the etiology of colorectal cancer. We are carrying out further laboratory studies to characterize the molecular mechanism underlying the risk associated with NATI genotypes. Additional epidemiological studies of colorectal cancer and other exposure-related cancers are in progress, and we are hopeful that these studies will substantiate our finding that NATI alÃ-elesare important genetic determinents of colorectal cancer risk.

presumably by increasing NATI mRNA levels. Considering these data with the present finding of risk for the NATl*]0 alÃ-ele,we suggest that the NATI enzyme may have an important role in the colon, possibly in activating carcinogens in the diet or in cigarette smoke. For example, NATI-mediated O-acetylation of W-hydroxy com pounds, or NATl-mediated MO-intramolecular acetyltransfer reac tions may result in activation of some arylamines or heterocylic amines; thus, high NATI activity could increase risk for some expo sures. The higher frequency of NAT'1*10 genotypes among smoking cases relative to nonsmoking cases (52 to 41%, respectively) suggests the possibility of an interaction between NATI and smoking exposure. We also observed that NAT1*10 genotypes were more common among patients with higher stage tumors. Exposure to carcinogens has been associated with advance stage in other cancer studies (26), and we suggest that the stage effect we observe may represent an inter action between NATI and exposure to carcinogens. That is, more advanced tumors have more genetic damage (27), and if this damage is, in part, caused by exposure to carcinogens, then NATI might be expected to have a greater effect in advanced stage tumors. In short, our observations in this study may be due to the NATI enzyme modulating the process of chemical carcinogenesis. Preliminary data from our laboratory indicate that the NATI* 10 alÃ-elealso predisposes heavy smokers to develop bladder cancer (28).5 Thus, in the present

ACKNOWLEDGMENTS We thank Drs. Jack Taylor and Ari Hirvoncn (National Institute of Envi ronmental Health Sciences) and Dr. Bob Millikan and Gary Pittman (University of North Carolina, Chapel Hill) for helpful comments on the manuscript.

REFERENCES 1. Potter, J. D., Slatery M. L., Bostick R. M., and Gapstur, S. M. Colon cancer: a review of the epidemiology. Epidemiol. Rev., 15: 499-545, 1993. 2. Giovannucci, E., Rimm, E. B., Stampfer, M, J., Hunter, D., Rosner, B., Willett, W. C, and Speizer, F. E. A prospective study of cigarette smoking and risk of colorectal adenoma and colorectal cancer in US men. J. Nati. Cancer Inst., 86: 183-191, 1994. 3. Vineis. P. Epidemiology of cancer from exposure to arylamines. Environ. Health Perspect., 706 (Suppl. 6): 7-10, 1994. 4. Felton, J. S., Knize, M., Dolbeare. F. A., and Wu, R. Mutagenic activity of hetercyclic amines in cooked foods. Environ. Health Perspect., 106 (Suppl. 6).- 201-204, 1994. 5. Fu, P. P., Herreno-Saenz, D., Von Tungeln, L. S., Lay, J. O., Wu, Y-S., Lai, J-S., and Evans, F. E. DNA adducts and carcinogenicity of nitro-polycyclic aromatic hydro carbons. Environ. Health Perspect., 106 (Suppl. 6): 177-184, 1994. 6. Hein, D. W., Doll, M. A., Rustan, T. D., Gray, K., Feng, Y., Ferguson, R. J.. and Grant, D. M. Metabolic activation and deactivation of arylamine carcinogens by recombinant human NATI and NAT2 acetyltransferases. Carcinogenesis (Lond.). 14: 1633-1638, 1993. 7. Blum, M., Demierre, A., Grant, D. M., Heim, M., and Meyer, U. A. Molecular mechanism of slow acetylation of drugs and carcinogens in humans. Proc. Nati. Acad. Sci. USA, 88: 5237-5241, 1991. 8. Deguchi, T., Mashimo, M., and Suzuki, T. Correlation between acetylator phenotypes and genotypes of polymorphic arylamine /V-acetyltransferase in human liver. J. Biol. Chem., 267: 18140-18147, 1990. 9. Vatsis, K. P., Marteli, K. J., and Weber, W. Diverse point mutations in the human gene for polymorphic N-acetyltransferase. Proc. Nati. Acad. Sci. USA, 88: 6333-6337, 1991. 10. Hickman, D., Risch, A., Camilleri, J., and Sim, E. Genotyping human polymorphic arylamine W-acetyltransferase: identification of new slow allotypic variants. Pharmacogenetics, 2: 217-226, 1992. 11. Bell, D. A., Taylor, J. A., Butler, M. A., Stephens. E., Wiest, J.. Brubaker, L, Kadlubar, F., and Lucier, G. W. Genotype/phenotype discordance for human aryl amine /V-acetyl transferase (NAT2) reveals a new slow-acetylator alÃ-elecommon in African-Americans. Carcinogenesis (Lond.), 14: 1689-1692, 1993. 12. Evans, D. P., /V-acetyltransferase. Pharmacol. Ther., 42: 157-234, 1989. 13. Lin H. J., Han, C-Y, Lin, B. K., and Hardy, S. Ethnic distribution of slow acetytator mutations in polymorphic /V-acetyltransferase (NAT2) gene. Pharmacogenetics, 4: 125-134, 1994. 14. Probst, M., Blum, M., Fasshauer, I., D'Orazio, D., Meyer, U., and Wild, D. The role

study it may be that either dietary or smoking-related carcinogens contribute to the association between rapid NATI genotypes and higher stage tumors. We also have preliminary data suggesting that the NATI*10 alÃ-elehas a similar association with advanced stage gastric adenocarcinoma (29);6 a finding that would be consistent with a role for NATI in some common exposure-related etiology for these two cancers. In contrast to the significant association observed between NATI rapid genotypes and colorectal cancer, we detect no significantly increased risk associated with NAT2 rapid acetylation genotypes. It has been proposed that carcinogens formed in the broiling and frying of meat are metabolized through an JV-hydroxylation step catalyzed by CYP1A2 that is followed by O-acetylation via acetyltransferases (either NATI or NAT2) (14, 20, 21, 30). This is believed to be an activation pathway for these compounds, and if so, the rapid acetylator genotype (NAT2) would be a risk factor among those that consume meats cooked by these methods (18-20). Several prelimi nary studies have been consistent with this hypothesis (18-20), al though a more detailed follow-up study suggests that NAT2 rapid acetylation conveys significant risk only in combination with rapid oxidation phenotype (CYP1A2) and consumption of well cooked meat (21). In addition, a recent epidemiological study of colorectal adenoma by Probst-Hensch et al. (31) failed to detect a significant role for NAT2 genotype. In that study, as in the present study, data on cooking method preference were not collected. This suggests that one cannot detect a main effect for NAT2 genotype unless data on other effect modifiers are available or the prevalence of exposure to dietary carcinogens derived from well cooked meats is very high among the study participants. It is interesting that the NATI gene effect was most apparent among those with the NAT2 rapid acetylator genotype (Table 5); unfortu nately, a mechanistic basis for the potential interaction between these

15. 16.

17.

5 }. A. Taylor, D. Umbach, E. Stephens, D. Paulson, C. Robertson, J. L. Mohler, and D. A. Bell. Role of W-acetylation polymorphism at NATI and NAT2 in smoking-associated bladder cancer. Manuscript in preparation. 6 D. A. Bell, M. Watson, T. Castranio, E. Stephens, M. Deakin, J. Elder, H. Duncan,

19.

C. Hendrickse, and R. C. Strange. A new genetic risk marker: NATI gene defect is associated with increased risk for gastric cancer. Manuscript in preparation.

20.

18.

of the human acetylation polymorphism in the metabolic activation of the food carcinogen 2-amino-3-methylimidazo[4,5-/]quinoline (10). Carcinogenesis (Lond.). 13: 1713-1717, 1992. Vatsis, K. P, and Weber, W. W. Structural heterogeneity of Caucasian W-acelyltrasferse at the NATI locus. Arch. Biochem. Biophys.. 301: 71-76, 1993. Grant, D. M., Vohra, P., Avis, Y., and Ima, A. Detection of a new polymorphism of human arylamine JV-acetyltransferase (NATI) using p-aminosalicylic acid as an in vivo probe (Abstract). J. Basic Clin. Physiol. Pharmacol., 3 (Suppl.).- 244, 1992. Weber, W. W., and Vatsis, K. P. Individual variability in p-aminobenzoic acid W-acetylation by human Af-acetyltrasferase (NATI) of peripheral blood. Pharmaco genetics, 3: 209-212, 1993. Wohlleb, J. C., Hunter, C. F., Blass, B., Kadlubar, F. F., Chu, D. Z., and Lang, N. P. Aromatic amine acetyltransferase as a marker for colorectal cancer: environmental and demographic associations. Int. J. Cancer, 46: 22-30, 1990. HeÂ«,K. F., David, B., Dethcon, P., Castleden, W., and Kwa, R. Acetylator phenotype in colorectal carcinoma. Cancer Res., 47: 1466-1469, 1991. Kadlubar, F., Butler, M. A., Kaderlik, K., Chou, H-C, and Lang, N. Polymorphisms

3541


NATI POLYMORPHISM

21.

22.

23.

24.

25.

AND COLORECTAL

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