- Email: [email protected]
Investigation of mutant frequency at the HPRT locus and changes in microsatellite sequences in healthy young adults
Mutation Research 431 Ž1999. 317–323 www.elsevier.comrlocatermolmut Community address: www.elsevier.comrlocatermutres
Investigation of mutant frequency at the HPRT locus and changes in microsatellite sequences in healthy young adults Margaret J. Davies ) , Joanne G. Turner, Cristofol Vives-Bauza, Paul C. Rumsby Department of Molecular Biology, BIBRA International, Woodmansterne Road, Carshalton, Surrey, SM5 4DS, UK Received 5 May 1999; received in revised form 30 August 1999; accepted 30 August 1999
Abstract In an attempt to understand the inter-individual variation that occurs in in vivo mutant frequency at the HPRT locus, we have examined the effect of polymorphisms in genes for metabolic enzymes on the mutation rate. In the same population of human volunteers, the background variant frequency in a number of microsatellite sequences was studied to determine individual variation in the capacity to repair mismatches in these sequences. The HPRT mutant frequency of T-cells isolated from a group of 49 healthy, non-smoking adults varied from 0.25 to 9.64 = 10y6 . The frequency of polymorphisms in CYP1A1, GSTM1 and NAT2 among these individuals was similar to those published, and when subjected to univariate analysis these polymorphisms showed no influence on the HPRT mutant frequency. However, there was a significant interaction between the GSTM1 null genotype and the slow acetylator status in NAT2 Ž P - 0.05. which was associated with higher mutant frequency. Analysis of 30 microsatellite sequences in 20 HPRT proficient clones per individual showed only six alterations in total, giving an overall mutation rate per allele of 0.01%, whilst three alterations were found in five HPRT deficient clones per individual examined for changes in 10 microsatellites, giving an overall mutation rate per allele of 0.3%. Thus, the alterations detected are probably due to background mutations and not to differences in mismatch repair capacity. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Human HPRT; Genetic polymorphisms; Microsatellites
1. Introduction The HPRT locus is frequently used to determine somatic mutation rates in the T-lymphocytes of populations exposed to known or suspected genotoxic agents and has proved useful in determining the mechanisms by which DNA damage occurs in human cells in vivo. However, the measurement of )
Corresponding author. Tel.: q44-181-652-1025; fax: q44181-661-7029. E-mail address: [email protected] ŽM.J. Davies.
human exposure to exogenous and endogenous genotoxins is confounded by the inter-individual variation that is observed in response to mutagenic insult. The range, which in normal individuals spans more than one order of magnitude w1–3x, often makes the interpretation of the results difficult and requires the study of large populations in order to determine an effect. This variation has been attributed, at least in part, to differences in the ability of individuals to activate or detoxify DNA-damaging substances and for some of the metabolic enzymes involved genetic polymorphisms have been implicated. Most previous
0027-5107r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 7 - 5 1 0 7 Ž 9 9 . 0 0 1 7 5 - X
318
M.J. DaÕies et al.r Mutation Research 431 (1999) 317–323
studies have been concerned with the influence of genetic polymorphisms on genotoxicity after occupational exposure w4,5x. However, these enzymes are also of importance in the metabolism of compounds associated with lifestyle factors such as diet, and therefore will also influence the background mutant frequency in populations not known to be exposed to a particular exogenous genotoxin. The cytochrome P450 gene, CYP1A1, is involved in the activation of polycyclic aromatic hydrocarbons found in the diet and cigarette smoke and polymorphisms in the gene have been linked to lung cancer w6x. The glutathione S-transferase Ž GSTM1. gene is concerned with the detoxification of both aromatic and heterocyclic amines which are formed during cooking, particularly of meats and their products w7x. The null GSTM1 genotype has been associated with increased risk of lung, stomach, colon and bladder cancer w8x. In addition certain vegetables and food additives are known to increase GST enzyme levels and, hence, potentiate the possible protective role of the wild-type gene w9x. A second phase II enzyme, N-acetyltransferase 2 Ž NAT2 ., deactivates aromatic amines by N-acetylation but activates heterocyclic amines by O-acetylation. The slow acetylator phenotype is associated with bladder cancer whilst the fast acetylator phenotype has been linked to colon cancer w10x. Another possible cause of inter-individual variation in mutation rates may be in the ability to repair DNA damage. Recently, much interest has been shown in the instability of microsatellite sequences and their association with cancer. Background mutation rates in microsatellites are of interest since it has been suggested that environmental agents may play a role in microsatellite changes w11x. Cell lines which are deficient in one of the mismatch repair genes and, hence, exhibiting increased microsatellite mutation frequencies also show an increased mutation rate at the HPRT locus w12x. In the present study, we have investigated the mutant frequency at the HPRT locus and analysed mutation in a number of microsatellite sequences of various repeated nucleotides in T-lymphocytes isolated from healthy, young, non-smoking adults. The influence of polymorphisms in the metabolism genes, CYP1A1, GSTM1 and NAT2 on HPRT mutant frequency was also determined in the study population.
2. Methods 2.1. Study population Twenty-five male and 24 female volunteers aged between 18 and 25 years were used for this study, none of whom were considered to be occupationally exposed to genotoxins. A questionnaire was used to obtain information on lifestyle factors which might contribute to the results. All participants were healthy and used only occasional medication such as antihistamines and headache remedies. X-ray exposure was limited to dental use and occasional sports injuries. The contraceptive pill was used by 48% of females. No volunteer currently smoked, although six females and four males had indulged at some point in the past five years. Samples of 50 ml blood were obtained by venepuncture into heparinised tubes from each participant. 2.2. Lymphocyte cloning Mononuclear cells were separated from blood by density gradient centrifugation and cloned as described previously w13x. Briefly, the cells were resuspended at 10 6 cells per ml of RPMI 1640 medium ŽDutch modification. supplemented with sodium pyruvate, L-glutamine, antibiotics and bovine serum and preincubated for 20 h. Non-adhering cells were harvested, the T-cell population counted and used to inoculate 96-well plates. Each well also contained 10 4 feeder cells and growth medium supplemented with interleukin-2 Ž20 Urml. and phytohaemaglutinin Ž5 mgrml.. Non-selective plates contained two cells per well, while selective plates for HPRT deficient mutants received 2 = 10 4 T-cells in medium containing 2.5 mgrml 6-thioguanine. The plates were incubated for 16 days before scoring. Clones from selective and non-selective plates were expanded for each individual to provide DNA for microsatellite analysis. 2.3. Polymorphism analysis NAT2 was analysed by the method of Hou et al. w4x. A 578-bp fragment of the intronless NAT2 gene was amplified by PCR before being separately digested with the restriction enzymes KpnI, BamHI
M.J. DaÕies et al.r Mutation Research 431 (1999) 317–323
and TaqI to detect the most common polymorphisms in this gene. The wild-type gene is cleaved at all three restriction sites, whereas a polymorphism in a restriction site results in the full length fragment being retained. GSTM1 was studied as described by Bell et al. w14x. Since the polymorphism results in a total deletion of the gene, a control PCR using a fragment of the HPRT gene was performed in the same reaction tube. CYP1A1 was analysed for the MspI polymorphism after PCR of a 340-bp fragment by the method of Hayashi et al. w15x. The isoleucine–valine poly-
319
morphism was detected by allele specific amplification as described by Sivaraman et al. w16x. Statistical analysis was carried out using the Minitab software package and analysis of variance ŽANOVA general linear model. to assess the effect of each individual polymorphism on mutant frequency. Possible interactions between the polymorphic genes were determined using the same model. 2.4. Microsatellite analysis Thirty microsatellite sequences were analysed for wild-type clones and 10 for the HPRT mutants; these are given in Table 1. Oligonucleotide primers
Table 1 Microsatellite sequences analysed Locus
Chromosome location
Microsatellite repeat
Polymorphic alleles
CRP GCG GLUT2 c-kit MCC Žexon 10. DPI ACTBP2 D7S440 LPL Žintron 6. ASS Žintron 14. D10S89 WT-1 INT-2 IGF-1 RB-1 D14S34 CYP19 SPN NF1 p53 Žintron 1. DCC DM SRC D21S198 CYP2DP8 HPRT
1q21–q23 2q36–q37 3q26 4q12 5q15–q23 5q15–q23 6 7q11 8p22 9q34 10p 11q13 11q13 12q23 13q14 14 15q25–qter 16q11.2 17q11.2 17p13.1 18q21 19q13 20q11.2 21q22.3 22q13 Xp26
9 8 14 – 4 13 21 10 3 10 4 – 9 11 – 5 – 18
AR AR TGFbR2 D-loop
Xq12 Xq12 3p mDNA
ŽCA.15 ŽGA. n ŽTA.16 ŽCA. 6 ŽTA.5 ŽCA. 9 ŽA. 25 ŽTG. 20 ŽCA.13 ŽAAAG.11 AAŽAAAG.15 ŽCA.19 ŽTTTA.10 ŽCA.16 ŽAC.10 AGŽAC. 21 A ŽGT. n ŽTG.5TCŽTG.16 ŽCT.16 ŽCTTTŽT.. 20 ŽCA.16 TAŽCA. 3 ŽCA. n ŽTC.11ŽAC.13 ŽGC.12 ŽCA. n ŽAAAAT. 8 ŽTA. 26 N28 ŽTA. 8 ŽGCT.5Žy 30. ŽCA. 20 ŽTG. 23 ŽGT.17 ŽGT. 9 ŽATGT. 3 ŽAT. 3 ŽGT. 6 ŽAT. 6 ŽAC. 2 AAACŽAT. 3 ACŽAT. 6 ŽAC. 3 AAŽAT.4 ACŽAT. 6 ŽAC. 7 ŽAT. 7 AGŽT.13 ŽCAG. 22 ŽGGC.16 ŽA.10 ŽCA.5
6 20 26 8 9 12 –
11 4 – 3
Control clones were analysed for changes in each of the 30 microsatellite sequences described above. HPRT mutants were examined for changes in the following microsatellites: DP1, MCC, ACTBP, LPL, IGF-1, SPN, NF1, p53, DM, TGFbR2.
M.J. DaÕies et al.r Mutation Research 431 (1999) 317–323
320 Table 2 Individual data for the donors Donor
24 11 22 9 14 15 20 21 1 5 6 25 13 7 3 26 10 27 16 17 12 23 28 31 46 35 50 49 29 53 32 2 34 52 41 37 45 56 58 59 57 39 54 61 60 38 40 51 19
Age
23 20 18 18 18 19 22 20 18 24 18 18 25 24 25 22 18 18 18 18 22 18 21 18 18 18 20 18 18 25 25 18 25 22 25 21 18 18 18 18 21 20 18 21 24 18 18 18 18
Sex
M F F M M M F M M M M F F F F F F M M F M M M M M M M M M M F M F F F F F F M M M F F F F F M F F
Plating efficiency
Mutant frequency
Po
%CE
Po
MFr10 6 cells
CYP1A1
Polymorphisms GSTM1
NAT2
59r96 59r96 49r96 53r96 60r96 40r96 54r96 62r96 60r96 47r96 62r96 51r96 37r96 46r96 45r96 41r96 47r96 45r96 39r96 42r96 39r96 60r96 35r96 34r96 31r96 47r96 38r96 42r96 40r96 34r96 26r96 49r96 72r96 54r96 41r96 36r96 31r96 43r96 44r96 38r96 39r96 31r96 49r96 48r96 46r96 57r96 51r96 45r96 46r96
24.34 24.34 33.63 29.70 23.50 43.77 28.77 21.86 23.50 35.71 21.86 31.63 47.67 36.79 37.88 42.54 35.71 37.88 45.04 41.33 45.04 23.50 50.45 51.90 56.52 35.71 46.34 41.33 43.77 51.90 65.31 33.63 14.38 28.77 42.54 49.04 56.52 40.16 39.01 46.34 45.04 56.52 33.63 34.66 36.79 26.07 31.63 37.88 36.79
467r480 458r480 467r480 474r480 469r480 460r480 466r480 471r480 477r480 470r480 477r480 476r480 461r480 468r480 464r480 472r480 280r288 476r480 469r480 473r480 470r480 476r480 466r480 470r480 459r480 470r480 471r480 479r480 476r480 472r480 452r480 466r480 473r480 479r480 464r480 471r480 466r480 476r480 468r480 469r480 463r480 456r480 478r480 465r480 465r480 469r480 475r480 461r480 470r480
5.6 9.6 4.1 2.1 4.9 4.9 5.1 4.3 1.3 2.9 1.4 1.3 4.2 3.4 4.5 2.0 3.9 1.1 2.6 1.8 2.3 1.8 2.9 2.0 4.0 2.9 2.0 0.3 1.0 1.6 4.6 4.4 5.1 0.4 4.0 1.9 2.6 1.0 3.2 2.5 4.0 4.5 0.6 4.6 4.3 4.4 1.7 5.3 2.9
qrq qrq qrq qrq qrq qry qrq qrq qrq qry qrq qrq qry qrq qry qrq qrq qry qrq qrq qrq qrq qrq qrq qrq qrq qrq qrq qrq qrq qry qrq qrq qrq qrq qrq qrq qrq qrq qry qrq qrq qry qrq qry qrq qrq qrq qrq
null null WT WT null WT null WT WT WT WT WT WT null null null null null WT null WT null WT WT null null null null null null WT WT WT null WT WT WT WT null null WT WT null WT WT WT null null WT
S F S S F S F F S F F F S F F F F S S F F F S S F S S S F S S S S S F S S F S S F S S F S S S F S
The cloning efficiency ŽCE. was calculated as: CE s yln PorX where Po is the proportion of negative wells and X is the average number of cells plated per well. Mutant frequency ŽMF. was calculated as: MF s CE selective platesrCE non-selective plates.
M.J. DaÕies et al.r Mutation Research 431 (1999) 317–323
321
were taken from published sequences, chosen to include a variety of nucleotide sequences and included at least one microsatellite from each chromosome. The PCR amplifications were carried out in the presence of 32 P-dCTP, after which aliquots of the reaction products were denatured and subjected to non-denaturing polyacrylamide gel electrophoresis at 128C, using a Stratotherm temperature controller. When compared to clones of the same individual loss of a band, or a change in band size, represented loss of heterozygosity or a mutation within the microsatellite respectively.
Table 4 Microsatellite alterations in T-lymphocytes
3. Results
study. However, there was a significant interaction Ž P - 0.05. between the GSTM1 null genotype Ž GSTM1yry ) and slow acetylator status Ž NAT2 srs .. No other genotype combinations were found to influence HPRT mutant frequency. Twenty wild-type clones from each individual were analysed for microsatellite mutations using 30 sequences, an overall total of 29,400 PCR fragments, or 600 per individual. Only six alterations were found in the microsatellites analysed ŽTable 4.. It is unlikely that these mutations occurred in vitro during clonal expansion since a mixed genotype would have resulted in extra bands being visible, which were not observed. One individual had a mutation in two different microsatellites in a single clone: the SPN fragment had lost one allele and the NF1 gene had a small deletion in one copy of the ŽCA. n repeat. Small deletions, presumably of a single repeat, were detected in the PCR fragments of LPL and MCC, whist ACTBP showed a small increase in size. One allele of NF1 was lost in a clone from one volunteer. Overall, the mutation rate per allele was only 0.01%. Five HPRT mutants from each individual were analysed for 10 microsatellite sequences, a total of 2450 fragments. A small deletion in one allele of the p53 gene fragment was seen in a clone from one volunteer and two clones from one person showed many bands of decreasing size in TGFb R2, which is a mononucleotide repeat of adenine. Thus, for the limited number of HPRT mutants analysed the overall mutation rate was 0.30%, although it was not shown that the two clones were not derived from the same parentage, which would reduce the mutation rate.
The HPRT mutant frequency varied between 0.25 and 9.64 = 10y6 ŽTable 2. and did not appear to be influenced by sex or any of the lifestyle factors recorded, such as contraceptive pill usage, alcohol, diet or exercise. Of the volunteers 18% were heterozygous for the CYP1A1 MspI polymorphism but no individual was homozygous for this mutation. The leucine–valine polymorphism of CYP1A1 was not observed in this population. Absence of both alleles of the GSTM1 gene occurred in 51% of the population. Polymorphisms in both alleles of the NAT2 gene were found in 57% of the volunteers which would result in the slow acetylator status in these individuals ŽTable 3.. These frequencies of polymorphisms are similar to those reported in the literature. None of these polymorphisms when analysed individually were found to influence the HPRT mutant frequency in this Table 3 NAT2 genotypes Genotype
No. individuals
Frequency
WILDTYPErWILDTYPE WILDTYPErKPN WILDTYPErTAQ WILDTYPErBAM KPNrKPN KPNrTAQ KPNrBAM TAQrTAQ TAQrBAM BAMrBAM
6 11 3 1 8 16 2 0 3 0
0.12 0.22 0.06 0.02 0.16 0.32 0.04 0 0.06 0
Individual
Clone
Microsatellite
Alteration
15 15 19 27 24 49 46 2 2
wild-type wild-type wild-type wild-type wild-type wild-type HPRT mutant HPRT mutant HPRT mutant
NF-1 SPN LPL MCC ACTBP NF-1 p53 TGFbR2 TGFbR2
small deletion loss of one band small deletion small deletion insertion loss of one band small deletion instability instability
322
M.J. DaÕies et al.r Mutation Research 431 (1999) 317–323
4. Discussion Factors which contribute to the variability in background mutant frequency at the HPRT locus are important when comparing control and exposed populations. Many investigations have shown age and smoking history contribute to this variation w1,17x. In the present study, we have used a population of healthy non-smokers, aged between 18 and 25 years in order to assess the influence of metabolic enzymes on mutant frequency. When analysed independently polymorphisms in the CYP1A1, GSTM1 and NAT2 genes were not found to influence HPRT mutant frequency. However, there was a significant interaction between the GSTM1 null genotype and the slow acetylator in NAT2 Ž p - 0.05. which was associated with higher mutant frequency. Other studies have also reported a relationship between the combination of polymorphisms in GSTM1 and NAT2 and HPRT mutant frequency in both non-exposed and exposed populations w4,18x. The low numbers of mutations in the microsatellite sequences analysed suggest that these mutations were not due to abnormalities in mismatch repair genes, but to background mutation in the loci examined. Hackman et al. w19x examined three microsatellite loci in 154 HPRT defective T-cell clones isolated from 28 individuals and found three size alterations in the fragments. The overall mutation rate determined by these authors was, at 0.29% per allele, very similar to that seen in the present study for HPRT mutants, at 0.30% per allele. However, the two clones from the donor which showed instability in the same locus may have derived from the same parentage. The mutation rate in HPRT proficient clones was lower at 0.01% per allele which might reflect the greater number of alleles investigated. Satoh et al. w20x found no size alterations when they analysed germline mutations in five tandem repeat sequences in 64 children from 50 families where at least one parent had been exposed to radiation Ž) 0.01 Sv., and only four alterations in a group of 60 children from 50 families where no parental radiation exposure had occurred. This suggests, as does our study, that these changes are due to the spontaneous mutation rate in the analysed loci and not to differences in the ability to repair mismatches. Li et al. w21x analysed alterations in seven tandem
repeats in the TK6 lymphoblast cell line and found three mutations in 193 TK proficient clones, two of which were in untreated cells. These authors also reported a total of 24 alterations in 331 TK deficient TK6 clones, 13 of these being in untreated cells. The frequency of microsatellite mutations in tumour cell lines varies considerably according to whether the cell line carries a mutation in one of the mismatch repair genes or not. In studies of colon cancer cell lines, the frequency of mutation per allele was found to vary between ) 0.3% in mismatch repair proficient lines and 23.0% in repair deficient cell w22x. In conclusion, this study has shown no effect of the status of the metabolising enzymes CYP1A1, GSTM1 and NAT2 when analysed independently on the mutant frequency at the HPRT locus in healthy, non-smoking, young adults. However, a relationship was evident between the GSTM1 null genotype and slow acetylator status which was associated with higher HPRT mutation rate. The low frequency of microsatellite changes detected showed that there were no differences between individuals in the ability to repair mismatches.
Acknowledgements This work was supported by the UK Ministry of Agriculture, Fisheries and Food. BIBRA was part of a EU concerted action project ŽBMH4–CT96–0120. entitled: Occupational and environmental mutagenesis: validation and application of the HPRT in vivo mutation assay for risk assessment in humans.
References w1x A.D. Tates, F.J. van Dam, H. van Mossel, H. Schoemaker, J.P.C. Thijssen, V.M. Woldring, A.H. Zwinderman, A.T. Natarajan, Use of the clonal assay for the measurement of frequencies of HPRT mutants in T-lymphocytes from five control populations, Mutat. Res. 253 Ž1991. 199–213. w2x R.F. Branda, L.M. Sullivan, J.P. O’Neill, M.T. Falta, J.A. Nicklas, B. Hirsch, P.M. Vacek, R.J. Albertini, Measurement of HPRT mutant frequencies in T-lymphocytes from healthy human populations, Mutat. Res. 285 Ž1993. 267–279. w3x J. Cole, T.R. Skopek, Somatic mutant frequency, mutation rates and mutational spectra in the human population in vivo, Mutat. Res. 304 Ž1994. 33–105. w4x S.-M. Hou, B. Lambert, K. Hemminki, Relationship between
M.J. DaÕies et al.r Mutation Research 431 (1999) 317–323
w5x
w6x
w7x w8x
w9x
w10x
w11x w12x
w13x
w14x
HPRT mutant frequency, aromatic DNA adducts and genotypes for GSTM1 and NAT2 in bus maintenance workers, Carcinogenesis 16 Ž1995. 1913–1917. R. Scarpato, L. Migliore, A. Hirvonen, G. Falck, H. Norppa, Cytogenetic monitoring of occupational exposure to pesticides: characterisation of GSTM1, GSTT1 and NAT2 genotypes, Environ. Molec. Mutagen. 27 Ž1996. 263–269. K. Kawajiri, K. Nakachi, K. Imai, A. Yoshii, N. Shinoda, J. Watanabe, Identification of genetically high risk individuals to lung cancer by polymorphisms of the cytochrome P4501A1 gene, FEBS Lett. 263 Ž1990. 131–133. K. Skog, Cooking procedures and food mutagens: a literature review, Food Chem. Toxicol. 31 Ž1993. 655–675. A. Puga, D.W. Nebert, R.A. McKinnon, A.G. Menon, Genetic polymorphisms in human drug-metabolising enzymes: potential uses of reverse genetics to identify genes of toxicological relevance, Crit. Rev. Toxicol. 27 Ž1997. 199–222. H. Verhagen, G. van Poppel, Short-term consumption of brussels sprouts leads to an increase in detoxifying glutathione-S-transferase enzymes and to a decrease in oxidative DNA-damage in human volunteers, in: M.J. Sadler, M. Saltmarsh ŽEds.., Functional Foods — The Consumer, the Products and the Evidence, Publ. R. Soc. Med. 1998, pp. 106–110. F.P. Perera, Molecular epidemiology: insights into cancer susceptibility, risk assessment and prevention, J. Natl. Cancer Inst. 88 Ž1996. 496–509. D. Holzman, Mismatch repair genes matched to several new roles in cancer, J. Natl. Cancer Inst. 88 Ž1996. 950–951. W.E. Glabb, K.R. Tindall, Mutation rate at the HPRT locus in human cancer cell lines with specific mismatch repair-gene defects, Carcinogenesis 18 Ž1997. 1–8. M.J. Davies, D.P. Lovell, D. Anderson, Thioguanine-resistance mutant frequency in T-lymphocytes from a healthy human population, Mutat. Res. 265 Ž1992. 165–171. D.A. Bell, J.A. Taylor, D.F. Paulson, C.N. Robertson, J.L.
w15x
w16x
w17x
w18x
w19x
w20x
w21x
w22x
323
Mohler, G.W. Lucier, Genetic risk and carcinogen exposure: a common inherited defect of the carcinogen-metabolism gene glutathione S-transferase M1 ŽGSTM1. that increases susceptibility to bladder cancer, J. Natl. Cancer Inst. 85 Ž1993. 1159–1164. S.-I. Hayashi, J. Watanabe, N. Nakacki, K. Kawajiri, Genetic linkage of lung cancer-associated MspI polymorphisms with amino acid replacement in the heme binding region of the human cytochrome P4501A1 gene, J. Biochem. 110 Ž1991. 407–411. L. Sivaraman, M.P. Leatham, J. Yee, L.R. Wilkins, A.F. Lau, L. Le Marchand, CYP1A1 genetic polymorphisms and in situ colorectal cancer, Cancer Res. 54 Ž1994. 3692–3695. J. Cole, M.H.L. Green, S.E. James, L. Henderson, H. Cole, A further assessment of factors influencing measurements of thioguanine-resistant mutant frequency in circulating Tlymphocytes, Mutat. Res. 204 Ž1988. 493–507. S.-M. Hou, S. Falt, ¨ A.-M. Steen, HPRT mutant frequency and GSTM1 genotype in non-smoking healthy individuals, Environ. Mol. Mutagen. 25 Ž1995. 97–105. ¨ P. Hackman, G. Gabbani, A.-M. Osterholm, D. Hellgren, B. Lambert, Spontaneous length variation in microsatellite DNA from human T-cell clones, Genes, Chromosomes Cancer 14 Ž1995. 1–5. C. Satoh, N. Takahashi, J.-I. Asakawa, M. Kodaira, R. Kuick, S.M. Hanash, J.V. Neel, Genetic analysis of children of atom bomb survivors, Environ. Health Perspect. 104 Ž1996. 511–519. C.-Y. Li, D.W. Yandell, J.B. Little, Elevated frequency of microsatellite mutations in TK6 human lymphocyte clones selected for mutations at the thymidine kinase locus, Molec. Cell. Biol. 14 Ž1994. 4373–4379. N. Bhattacharyya, A. Skandalis, A. Ganesh, J. Groden, M. Meuth, Mutator phenotypes in colorectal carcinoma cell lines, Proc. Natl. Acad. Sci. USA. 91 Ž1994. 6319–6323.
Investigation of mutant frequency at the HPRT locus and changes in microsatellite sequences in healthy young adults Margaret J. Davies ) , Joanne G. Turner, Cristofol Vives-Bauza, Paul C. Rumsby Department of Molecular Biology, BIBRA International, Woodmansterne Road, Carshalton, Surrey, SM5 4DS, UK Received 5 May 1999; received in revised form 30 August 1999; accepted 30 August 1999
Abstract In an attempt to understand the inter-individual variation that occurs in in vivo mutant frequency at the HPRT locus, we have examined the effect of polymorphisms in genes for metabolic enzymes on the mutation rate. In the same population of human volunteers, the background variant frequency in a number of microsatellite sequences was studied to determine individual variation in the capacity to repair mismatches in these sequences. The HPRT mutant frequency of T-cells isolated from a group of 49 healthy, non-smoking adults varied from 0.25 to 9.64 = 10y6 . The frequency of polymorphisms in CYP1A1, GSTM1 and NAT2 among these individuals was similar to those published, and when subjected to univariate analysis these polymorphisms showed no influence on the HPRT mutant frequency. However, there was a significant interaction between the GSTM1 null genotype and the slow acetylator status in NAT2 Ž P - 0.05. which was associated with higher mutant frequency. Analysis of 30 microsatellite sequences in 20 HPRT proficient clones per individual showed only six alterations in total, giving an overall mutation rate per allele of 0.01%, whilst three alterations were found in five HPRT deficient clones per individual examined for changes in 10 microsatellites, giving an overall mutation rate per allele of 0.3%. Thus, the alterations detected are probably due to background mutations and not to differences in mismatch repair capacity. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Human HPRT; Genetic polymorphisms; Microsatellites
1. Introduction The HPRT locus is frequently used to determine somatic mutation rates in the T-lymphocytes of populations exposed to known or suspected genotoxic agents and has proved useful in determining the mechanisms by which DNA damage occurs in human cells in vivo. However, the measurement of )
Corresponding author. Tel.: q44-181-652-1025; fax: q44181-661-7029. E-mail address: [email protected] ŽM.J. Davies.
human exposure to exogenous and endogenous genotoxins is confounded by the inter-individual variation that is observed in response to mutagenic insult. The range, which in normal individuals spans more than one order of magnitude w1–3x, often makes the interpretation of the results difficult and requires the study of large populations in order to determine an effect. This variation has been attributed, at least in part, to differences in the ability of individuals to activate or detoxify DNA-damaging substances and for some of the metabolic enzymes involved genetic polymorphisms have been implicated. Most previous
0027-5107r99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 7 - 5 1 0 7 Ž 9 9 . 0 0 1 7 5 - X
318
M.J. DaÕies et al.r Mutation Research 431 (1999) 317–323
studies have been concerned with the influence of genetic polymorphisms on genotoxicity after occupational exposure w4,5x. However, these enzymes are also of importance in the metabolism of compounds associated with lifestyle factors such as diet, and therefore will also influence the background mutant frequency in populations not known to be exposed to a particular exogenous genotoxin. The cytochrome P450 gene, CYP1A1, is involved in the activation of polycyclic aromatic hydrocarbons found in the diet and cigarette smoke and polymorphisms in the gene have been linked to lung cancer w6x. The glutathione S-transferase Ž GSTM1. gene is concerned with the detoxification of both aromatic and heterocyclic amines which are formed during cooking, particularly of meats and their products w7x. The null GSTM1 genotype has been associated with increased risk of lung, stomach, colon and bladder cancer w8x. In addition certain vegetables and food additives are known to increase GST enzyme levels and, hence, potentiate the possible protective role of the wild-type gene w9x. A second phase II enzyme, N-acetyltransferase 2 Ž NAT2 ., deactivates aromatic amines by N-acetylation but activates heterocyclic amines by O-acetylation. The slow acetylator phenotype is associated with bladder cancer whilst the fast acetylator phenotype has been linked to colon cancer w10x. Another possible cause of inter-individual variation in mutation rates may be in the ability to repair DNA damage. Recently, much interest has been shown in the instability of microsatellite sequences and their association with cancer. Background mutation rates in microsatellites are of interest since it has been suggested that environmental agents may play a role in microsatellite changes w11x. Cell lines which are deficient in one of the mismatch repair genes and, hence, exhibiting increased microsatellite mutation frequencies also show an increased mutation rate at the HPRT locus w12x. In the present study, we have investigated the mutant frequency at the HPRT locus and analysed mutation in a number of microsatellite sequences of various repeated nucleotides in T-lymphocytes isolated from healthy, young, non-smoking adults. The influence of polymorphisms in the metabolism genes, CYP1A1, GSTM1 and NAT2 on HPRT mutant frequency was also determined in the study population.
2. Methods 2.1. Study population Twenty-five male and 24 female volunteers aged between 18 and 25 years were used for this study, none of whom were considered to be occupationally exposed to genotoxins. A questionnaire was used to obtain information on lifestyle factors which might contribute to the results. All participants were healthy and used only occasional medication such as antihistamines and headache remedies. X-ray exposure was limited to dental use and occasional sports injuries. The contraceptive pill was used by 48% of females. No volunteer currently smoked, although six females and four males had indulged at some point in the past five years. Samples of 50 ml blood were obtained by venepuncture into heparinised tubes from each participant. 2.2. Lymphocyte cloning Mononuclear cells were separated from blood by density gradient centrifugation and cloned as described previously w13x. Briefly, the cells were resuspended at 10 6 cells per ml of RPMI 1640 medium ŽDutch modification. supplemented with sodium pyruvate, L-glutamine, antibiotics and bovine serum and preincubated for 20 h. Non-adhering cells were harvested, the T-cell population counted and used to inoculate 96-well plates. Each well also contained 10 4 feeder cells and growth medium supplemented with interleukin-2 Ž20 Urml. and phytohaemaglutinin Ž5 mgrml.. Non-selective plates contained two cells per well, while selective plates for HPRT deficient mutants received 2 = 10 4 T-cells in medium containing 2.5 mgrml 6-thioguanine. The plates were incubated for 16 days before scoring. Clones from selective and non-selective plates were expanded for each individual to provide DNA for microsatellite analysis. 2.3. Polymorphism analysis NAT2 was analysed by the method of Hou et al. w4x. A 578-bp fragment of the intronless NAT2 gene was amplified by PCR before being separately digested with the restriction enzymes KpnI, BamHI
M.J. DaÕies et al.r Mutation Research 431 (1999) 317–323
and TaqI to detect the most common polymorphisms in this gene. The wild-type gene is cleaved at all three restriction sites, whereas a polymorphism in a restriction site results in the full length fragment being retained. GSTM1 was studied as described by Bell et al. w14x. Since the polymorphism results in a total deletion of the gene, a control PCR using a fragment of the HPRT gene was performed in the same reaction tube. CYP1A1 was analysed for the MspI polymorphism after PCR of a 340-bp fragment by the method of Hayashi et al. w15x. The isoleucine–valine poly-
319
morphism was detected by allele specific amplification as described by Sivaraman et al. w16x. Statistical analysis was carried out using the Minitab software package and analysis of variance ŽANOVA general linear model. to assess the effect of each individual polymorphism on mutant frequency. Possible interactions between the polymorphic genes were determined using the same model. 2.4. Microsatellite analysis Thirty microsatellite sequences were analysed for wild-type clones and 10 for the HPRT mutants; these are given in Table 1. Oligonucleotide primers
Table 1 Microsatellite sequences analysed Locus
Chromosome location
Microsatellite repeat
Polymorphic alleles
CRP GCG GLUT2 c-kit MCC Žexon 10. DPI ACTBP2 D7S440 LPL Žintron 6. ASS Žintron 14. D10S89 WT-1 INT-2 IGF-1 RB-1 D14S34 CYP19 SPN NF1 p53 Žintron 1. DCC DM SRC D21S198 CYP2DP8 HPRT
1q21–q23 2q36–q37 3q26 4q12 5q15–q23 5q15–q23 6 7q11 8p22 9q34 10p 11q13 11q13 12q23 13q14 14 15q25–qter 16q11.2 17q11.2 17p13.1 18q21 19q13 20q11.2 21q22.3 22q13 Xp26
9 8 14 – 4 13 21 10 3 10 4 – 9 11 – 5 – 18
AR AR TGFbR2 D-loop
Xq12 Xq12 3p mDNA
ŽCA.15 ŽGA. n ŽTA.16 ŽCA. 6 ŽTA.5 ŽCA. 9 ŽA. 25 ŽTG. 20 ŽCA.13 ŽAAAG.11 AAŽAAAG.15 ŽCA.19 ŽTTTA.10 ŽCA.16 ŽAC.10 AGŽAC. 21 A ŽGT. n ŽTG.5TCŽTG.16 ŽCT.16 ŽCTTTŽT.. 20 ŽCA.16 TAŽCA. 3 ŽCA. n ŽTC.11ŽAC.13 ŽGC.12 ŽCA. n ŽAAAAT. 8 ŽTA. 26 N28 ŽTA. 8 ŽGCT.5Žy 30. ŽCA. 20 ŽTG. 23 ŽGT.17 ŽGT. 9 ŽATGT. 3 ŽAT. 3 ŽGT. 6 ŽAT. 6 ŽAC. 2 AAACŽAT. 3 ACŽAT. 6 ŽAC. 3 AAŽAT.4 ACŽAT. 6 ŽAC. 7 ŽAT. 7 AGŽT.13 ŽCAG. 22 ŽGGC.16 ŽA.10 ŽCA.5
6 20 26 8 9 12 –
11 4 – 3
Control clones were analysed for changes in each of the 30 microsatellite sequences described above. HPRT mutants were examined for changes in the following microsatellites: DP1, MCC, ACTBP, LPL, IGF-1, SPN, NF1, p53, DM, TGFbR2.
M.J. DaÕies et al.r Mutation Research 431 (1999) 317–323
320 Table 2 Individual data for the donors Donor
24 11 22 9 14 15 20 21 1 5 6 25 13 7 3 26 10 27 16 17 12 23 28 31 46 35 50 49 29 53 32 2 34 52 41 37 45 56 58 59 57 39 54 61 60 38 40 51 19
Age
23 20 18 18 18 19 22 20 18 24 18 18 25 24 25 22 18 18 18 18 22 18 21 18 18 18 20 18 18 25 25 18 25 22 25 21 18 18 18 18 21 20 18 21 24 18 18 18 18
Sex
M F F M M M F M M M M F F F F F F M M F M M M M M M M M M M F M F F F F F F M M M F F F F F M F F
Plating efficiency
Mutant frequency
Po
%CE
Po
MFr10 6 cells
CYP1A1
Polymorphisms GSTM1
NAT2
59r96 59r96 49r96 53r96 60r96 40r96 54r96 62r96 60r96 47r96 62r96 51r96 37r96 46r96 45r96 41r96 47r96 45r96 39r96 42r96 39r96 60r96 35r96 34r96 31r96 47r96 38r96 42r96 40r96 34r96 26r96 49r96 72r96 54r96 41r96 36r96 31r96 43r96 44r96 38r96 39r96 31r96 49r96 48r96 46r96 57r96 51r96 45r96 46r96
24.34 24.34 33.63 29.70 23.50 43.77 28.77 21.86 23.50 35.71 21.86 31.63 47.67 36.79 37.88 42.54 35.71 37.88 45.04 41.33 45.04 23.50 50.45 51.90 56.52 35.71 46.34 41.33 43.77 51.90 65.31 33.63 14.38 28.77 42.54 49.04 56.52 40.16 39.01 46.34 45.04 56.52 33.63 34.66 36.79 26.07 31.63 37.88 36.79
467r480 458r480 467r480 474r480 469r480 460r480 466r480 471r480 477r480 470r480 477r480 476r480 461r480 468r480 464r480 472r480 280r288 476r480 469r480 473r480 470r480 476r480 466r480 470r480 459r480 470r480 471r480 479r480 476r480 472r480 452r480 466r480 473r480 479r480 464r480 471r480 466r480 476r480 468r480 469r480 463r480 456r480 478r480 465r480 465r480 469r480 475r480 461r480 470r480
5.6 9.6 4.1 2.1 4.9 4.9 5.1 4.3 1.3 2.9 1.4 1.3 4.2 3.4 4.5 2.0 3.9 1.1 2.6 1.8 2.3 1.8 2.9 2.0 4.0 2.9 2.0 0.3 1.0 1.6 4.6 4.4 5.1 0.4 4.0 1.9 2.6 1.0 3.2 2.5 4.0 4.5 0.6 4.6 4.3 4.4 1.7 5.3 2.9
qrq qrq qrq qrq qrq qry qrq qrq qrq qry qrq qrq qry qrq qry qrq qrq qry qrq qrq qrq qrq qrq qrq qrq qrq qrq qrq qrq qrq qry qrq qrq qrq qrq qrq qrq qrq qrq qry qrq qrq qry qrq qry qrq qrq qrq qrq
null null WT WT null WT null WT WT WT WT WT WT null null null null null WT null WT null WT WT null null null null null null WT WT WT null WT WT WT WT null null WT WT null WT WT WT null null WT
S F S S F S F F S F F F S F F F F S S F F F S S F S S S F S S S S S F S S F S S F S S F S S S F S
The cloning efficiency ŽCE. was calculated as: CE s yln PorX where Po is the proportion of negative wells and X is the average number of cells plated per well. Mutant frequency ŽMF. was calculated as: MF s CE selective platesrCE non-selective plates.
M.J. DaÕies et al.r Mutation Research 431 (1999) 317–323
321
were taken from published sequences, chosen to include a variety of nucleotide sequences and included at least one microsatellite from each chromosome. The PCR amplifications were carried out in the presence of 32 P-dCTP, after which aliquots of the reaction products were denatured and subjected to non-denaturing polyacrylamide gel electrophoresis at 128C, using a Stratotherm temperature controller. When compared to clones of the same individual loss of a band, or a change in band size, represented loss of heterozygosity or a mutation within the microsatellite respectively.
Table 4 Microsatellite alterations in T-lymphocytes
3. Results
study. However, there was a significant interaction Ž P - 0.05. between the GSTM1 null genotype Ž GSTM1yry ) and slow acetylator status Ž NAT2 srs .. No other genotype combinations were found to influence HPRT mutant frequency. Twenty wild-type clones from each individual were analysed for microsatellite mutations using 30 sequences, an overall total of 29,400 PCR fragments, or 600 per individual. Only six alterations were found in the microsatellites analysed ŽTable 4.. It is unlikely that these mutations occurred in vitro during clonal expansion since a mixed genotype would have resulted in extra bands being visible, which were not observed. One individual had a mutation in two different microsatellites in a single clone: the SPN fragment had lost one allele and the NF1 gene had a small deletion in one copy of the ŽCA. n repeat. Small deletions, presumably of a single repeat, were detected in the PCR fragments of LPL and MCC, whist ACTBP showed a small increase in size. One allele of NF1 was lost in a clone from one volunteer. Overall, the mutation rate per allele was only 0.01%. Five HPRT mutants from each individual were analysed for 10 microsatellite sequences, a total of 2450 fragments. A small deletion in one allele of the p53 gene fragment was seen in a clone from one volunteer and two clones from one person showed many bands of decreasing size in TGFb R2, which is a mononucleotide repeat of adenine. Thus, for the limited number of HPRT mutants analysed the overall mutation rate was 0.30%, although it was not shown that the two clones were not derived from the same parentage, which would reduce the mutation rate.
The HPRT mutant frequency varied between 0.25 and 9.64 = 10y6 ŽTable 2. and did not appear to be influenced by sex or any of the lifestyle factors recorded, such as contraceptive pill usage, alcohol, diet or exercise. Of the volunteers 18% were heterozygous for the CYP1A1 MspI polymorphism but no individual was homozygous for this mutation. The leucine–valine polymorphism of CYP1A1 was not observed in this population. Absence of both alleles of the GSTM1 gene occurred in 51% of the population. Polymorphisms in both alleles of the NAT2 gene were found in 57% of the volunteers which would result in the slow acetylator status in these individuals ŽTable 3.. These frequencies of polymorphisms are similar to those reported in the literature. None of these polymorphisms when analysed individually were found to influence the HPRT mutant frequency in this Table 3 NAT2 genotypes Genotype
No. individuals
Frequency
WILDTYPErWILDTYPE WILDTYPErKPN WILDTYPErTAQ WILDTYPErBAM KPNrKPN KPNrTAQ KPNrBAM TAQrTAQ TAQrBAM BAMrBAM
6 11 3 1 8 16 2 0 3 0
0.12 0.22 0.06 0.02 0.16 0.32 0.04 0 0.06 0
Individual
Clone
Microsatellite
Alteration
15 15 19 27 24 49 46 2 2
wild-type wild-type wild-type wild-type wild-type wild-type HPRT mutant HPRT mutant HPRT mutant
NF-1 SPN LPL MCC ACTBP NF-1 p53 TGFbR2 TGFbR2
small deletion loss of one band small deletion small deletion insertion loss of one band small deletion instability instability
322
M.J. DaÕies et al.r Mutation Research 431 (1999) 317–323
4. Discussion Factors which contribute to the variability in background mutant frequency at the HPRT locus are important when comparing control and exposed populations. Many investigations have shown age and smoking history contribute to this variation w1,17x. In the present study, we have used a population of healthy non-smokers, aged between 18 and 25 years in order to assess the influence of metabolic enzymes on mutant frequency. When analysed independently polymorphisms in the CYP1A1, GSTM1 and NAT2 genes were not found to influence HPRT mutant frequency. However, there was a significant interaction between the GSTM1 null genotype and the slow acetylator in NAT2 Ž p - 0.05. which was associated with higher mutant frequency. Other studies have also reported a relationship between the combination of polymorphisms in GSTM1 and NAT2 and HPRT mutant frequency in both non-exposed and exposed populations w4,18x. The low numbers of mutations in the microsatellite sequences analysed suggest that these mutations were not due to abnormalities in mismatch repair genes, but to background mutation in the loci examined. Hackman et al. w19x examined three microsatellite loci in 154 HPRT defective T-cell clones isolated from 28 individuals and found three size alterations in the fragments. The overall mutation rate determined by these authors was, at 0.29% per allele, very similar to that seen in the present study for HPRT mutants, at 0.30% per allele. However, the two clones from the donor which showed instability in the same locus may have derived from the same parentage. The mutation rate in HPRT proficient clones was lower at 0.01% per allele which might reflect the greater number of alleles investigated. Satoh et al. w20x found no size alterations when they analysed germline mutations in five tandem repeat sequences in 64 children from 50 families where at least one parent had been exposed to radiation Ž) 0.01 Sv., and only four alterations in a group of 60 children from 50 families where no parental radiation exposure had occurred. This suggests, as does our study, that these changes are due to the spontaneous mutation rate in the analysed loci and not to differences in the ability to repair mismatches. Li et al. w21x analysed alterations in seven tandem
repeats in the TK6 lymphoblast cell line and found three mutations in 193 TK proficient clones, two of which were in untreated cells. These authors also reported a total of 24 alterations in 331 TK deficient TK6 clones, 13 of these being in untreated cells. The frequency of microsatellite mutations in tumour cell lines varies considerably according to whether the cell line carries a mutation in one of the mismatch repair genes or not. In studies of colon cancer cell lines, the frequency of mutation per allele was found to vary between ) 0.3% in mismatch repair proficient lines and 23.0% in repair deficient cell w22x. In conclusion, this study has shown no effect of the status of the metabolising enzymes CYP1A1, GSTM1 and NAT2 when analysed independently on the mutant frequency at the HPRT locus in healthy, non-smoking, young adults. However, a relationship was evident between the GSTM1 null genotype and slow acetylator status which was associated with higher HPRT mutation rate. The low frequency of microsatellite changes detected showed that there were no differences between individuals in the ability to repair mismatches.
Acknowledgements This work was supported by the UK Ministry of Agriculture, Fisheries and Food. BIBRA was part of a EU concerted action project ŽBMH4–CT96–0120. entitled: Occupational and environmental mutagenesis: validation and application of the HPRT in vivo mutation assay for risk assessment in humans.
References w1x A.D. Tates, F.J. van Dam, H. van Mossel, H. Schoemaker, J.P.C. Thijssen, V.M. Woldring, A.H. Zwinderman, A.T. Natarajan, Use of the clonal assay for the measurement of frequencies of HPRT mutants in T-lymphocytes from five control populations, Mutat. Res. 253 Ž1991. 199–213. w2x R.F. Branda, L.M. Sullivan, J.P. O’Neill, M.T. Falta, J.A. Nicklas, B. Hirsch, P.M. Vacek, R.J. Albertini, Measurement of HPRT mutant frequencies in T-lymphocytes from healthy human populations, Mutat. Res. 285 Ž1993. 267–279. w3x J. Cole, T.R. Skopek, Somatic mutant frequency, mutation rates and mutational spectra in the human population in vivo, Mutat. Res. 304 Ž1994. 33–105. w4x S.-M. Hou, B. Lambert, K. Hemminki, Relationship between
M.J. DaÕies et al.r Mutation Research 431 (1999) 317–323
w5x
w6x
w7x w8x
w9x
w10x
w11x w12x
w13x
w14x
HPRT mutant frequency, aromatic DNA adducts and genotypes for GSTM1 and NAT2 in bus maintenance workers, Carcinogenesis 16 Ž1995. 1913–1917. R. Scarpato, L. Migliore, A. Hirvonen, G. Falck, H. Norppa, Cytogenetic monitoring of occupational exposure to pesticides: characterisation of GSTM1, GSTT1 and NAT2 genotypes, Environ. Molec. Mutagen. 27 Ž1996. 263–269. K. Kawajiri, K. Nakachi, K. Imai, A. Yoshii, N. Shinoda, J. Watanabe, Identification of genetically high risk individuals to lung cancer by polymorphisms of the cytochrome P4501A1 gene, FEBS Lett. 263 Ž1990. 131–133. K. Skog, Cooking procedures and food mutagens: a literature review, Food Chem. Toxicol. 31 Ž1993. 655–675. A. Puga, D.W. Nebert, R.A. McKinnon, A.G. Menon, Genetic polymorphisms in human drug-metabolising enzymes: potential uses of reverse genetics to identify genes of toxicological relevance, Crit. Rev. Toxicol. 27 Ž1997. 199–222. H. Verhagen, G. van Poppel, Short-term consumption of brussels sprouts leads to an increase in detoxifying glutathione-S-transferase enzymes and to a decrease in oxidative DNA-damage in human volunteers, in: M.J. Sadler, M. Saltmarsh ŽEds.., Functional Foods — The Consumer, the Products and the Evidence, Publ. R. Soc. Med. 1998, pp. 106–110. F.P. Perera, Molecular epidemiology: insights into cancer susceptibility, risk assessment and prevention, J. Natl. Cancer Inst. 88 Ž1996. 496–509. D. Holzman, Mismatch repair genes matched to several new roles in cancer, J. Natl. Cancer Inst. 88 Ž1996. 950–951. W.E. Glabb, K.R. Tindall, Mutation rate at the HPRT locus in human cancer cell lines with specific mismatch repair-gene defects, Carcinogenesis 18 Ž1997. 1–8. M.J. Davies, D.P. Lovell, D. Anderson, Thioguanine-resistance mutant frequency in T-lymphocytes from a healthy human population, Mutat. Res. 265 Ž1992. 165–171. D.A. Bell, J.A. Taylor, D.F. Paulson, C.N. Robertson, J.L.
w15x
w16x
w17x
w18x
w19x
w20x
w21x
w22x
323
Mohler, G.W. Lucier, Genetic risk and carcinogen exposure: a common inherited defect of the carcinogen-metabolism gene glutathione S-transferase M1 ŽGSTM1. that increases susceptibility to bladder cancer, J. Natl. Cancer Inst. 85 Ž1993. 1159–1164. S.-I. Hayashi, J. Watanabe, N. Nakacki, K. Kawajiri, Genetic linkage of lung cancer-associated MspI polymorphisms with amino acid replacement in the heme binding region of the human cytochrome P4501A1 gene, J. Biochem. 110 Ž1991. 407–411. L. Sivaraman, M.P. Leatham, J. Yee, L.R. Wilkins, A.F. Lau, L. Le Marchand, CYP1A1 genetic polymorphisms and in situ colorectal cancer, Cancer Res. 54 Ž1994. 3692–3695. J. Cole, M.H.L. Green, S.E. James, L. Henderson, H. Cole, A further assessment of factors influencing measurements of thioguanine-resistant mutant frequency in circulating Tlymphocytes, Mutat. Res. 204 Ž1988. 493–507. S.-M. Hou, S. Falt, ¨ A.-M. Steen, HPRT mutant frequency and GSTM1 genotype in non-smoking healthy individuals, Environ. Mol. Mutagen. 25 Ž1995. 97–105. ¨ P. Hackman, G. Gabbani, A.-M. Osterholm, D. Hellgren, B. Lambert, Spontaneous length variation in microsatellite DNA from human T-cell clones, Genes, Chromosomes Cancer 14 Ž1995. 1–5. C. Satoh, N. Takahashi, J.-I. Asakawa, M. Kodaira, R. Kuick, S.M. Hanash, J.V. Neel, Genetic analysis of children of atom bomb survivors, Environ. Health Perspect. 104 Ž1996. 511–519. C.-Y. Li, D.W. Yandell, J.B. Little, Elevated frequency of microsatellite mutations in TK6 human lymphocyte clones selected for mutations at the thymidine kinase locus, Molec. Cell. Biol. 14 Ž1994. 4373–4379. N. Bhattacharyya, A. Skandalis, A. Ganesh, J. Groden, M. Meuth, Mutator phenotypes in colorectal carcinoma cell lines, Proc. Natl. Acad. Sci. USA. 91 Ž1994. 6319–6323.