of the NAT2 genotypes and the predicted phenotype in centenar- ian and control populations. Genotype definition: WT/WT, homozygote wild-type; WT/M1–M4, heterozygote; M1–M4/M1–M4, homozygote mutant. Only the observed geno- types are reported. Phenotype definitions deduced from combi- nations of NAT2 genotypes: slow acetylator, homozygote mutants; fast acetylator, genotype with one or two wild-type alleles

NAT2 genotypes Controls Centenarians n Frequency n Frequency NAT2*4/NAT2*4 0 0 3 0.025 NAT2*4/NAT2*5A 2 0.023 5 0.042 NAT2*4/NAT2*6A 8 0.091 5 0.042 NAT2*4/NAT2*7A 1 0.011 0 0 NAT2*5A/NAT2*5A 24 0.273 39 0.331 NAT2*5A/NAT2*6A 41 0.466 36 0.305 NAT2*5A/NAT2*7A 2 0.023 6 0.051 NAT2*6A/NAT2*6A 9 0.102 19 0.161 NAT2*6A/NAT2*7A 1 0.011 5 0.042 NAT2 predicted phenotypes Slow acetylator 77 0.875 105 0.890 Fast acetylator 11 0.125 13 0.110 Total 88 1 118 1

cant. The proportion of the GSTM1*0/GSTM1*0 genotype in the centenarian population (52.7%; n = 565) was not significantely different from that observed in controls (49.3%; n = 229).

Table 4 Distribution of the GSTM1 genotypes in centenarian and control populations (–GSTM1 gene absent, + GSTM1 gene detected)

GSTM1 gene Controls Centenarians n Frequency n Frequency – 113 0.493 298 0.527 + 116 0.507 267 0.473 Total 229 1 565 1

Predicted phenotype combinations

When the study populations were analyzed according to the combined predicted phenotype based on the knowl- edge of the genotypes (Table 5), 60 and 110 combinations were available for the control and the centenarian popula- tions, respectively. Of the controls, 50% were character- ized by the following combinations: CYP2D6/EM; NAT2/slow; GSTM1/present. The second major haplotype was identical except for the fact that it included the GSTM1 null version. By comparing the distribution of haplotypes observed at the CYP2D6, NAT2, and GSTM1 loci for the polymorphisms tested, we found no significant

(27.3% and 33.1%, respectively, for control and centenari- an populations) and NAT2*6A/*6A (46.6% and 30.5%). However, no significant difference between the two popu- lations was found as far as allele and genotype distribu- tions were concerned. Mutant alleles are known to be as- sociated with the slow phenotype when present without the wild-type allele, while the fast phenotype includes ho- mozygotes for the wild-type allele or heterozygotes with one wild-type and one of the mutant alleles. As shown in Table 3, results of 88 genotypes from control subjects and 118 from centenarians predicted a frequency of slow phe- notypes of 87.5% and 89%, respectively. These frequen- cies are clearly higher than those described in previous studies on other Caucasian populations in which the fre- quency of the slow acetylator phenotype is roughly 50% in Caucasians (Hickman and Sim 1991; Bell et al. 1993). However, these unexpected proportions were identical in the two populations tested, i.e., no difference could be found between centenarian and control populations as far as phenotype distributions were concerned.

Discussion

A number of independent studies has demonstrated that genetic polymorphisms in the metabolism of xenobiotics are risk factors in the development of diseases associated with environmental and chemical exposure. Since environ- mental and genetic factors must be considered when studying human aging and longevity, and since drug-me- tabolizing enzymes are of particular relevance in toxification and detoxification, we asked whether or not three of these polymorphic enzymes could account, alone or in combination, for one part of the genetic component of human longevity.

GSTM1 locus analysis

Table 4 shows the distribution of the GSTM1 genotypes detected in the two study groups. The frequency of the GSTM1 null genotype among our control subjects (49.3%) was slightly higher than earlier genotyping studies in other Caucasian populations (40–45%) (Zhong et al. 1993b; Hirvonen et al. 1995) but the difference was not signifi-

We selected a study population of French Caucasian centenarians (i.e., French Caucasians who were already proved to be long-lived) and controls (adult individuals aged 20–70 years). Of course, individual life expectancies of the controls are not known, and some controls in the fu-

Table 5 Distribution of combinations of CYP2D6, NAT2, and GSTM1 predicted phenotypes in centenarian and control populations

Phenotypes Controls Centenarians CYP2D6 NAT2 GSTM1 n Frequency n Frequency Poor metabolizer Slow acetylator Deficient 1 0.017 1 0.009 Active 0 0 3 0.027 Fast acetylator Deficient 0 0 1 0.009 Active 0 0 0 0 Extensive metabolizer Slow acetylator Deficient 23 0.383 46 0.418 Active 30 0.500 47 0.427 Fast acatylator Deficient 2 0.033 6 0.055 Active 4 0.067 6 0.055 Total 60 1 110 1

very valuable in the case of studies on very old people as centenarians, given the difficulties in obtaining multiple biological samples. Agundez et al. (1997) recently studied CYP2D6, NAT2, and CYP2E1 genetic polymorphisms in 41 Spanish nonagenarians and failed to detect any rela- tionship between any configuration of these three poly- morphic traits and longevity. Their results and ours, ob- served on a larger population of centenarians, strengthen the hypothesis that genetically determined differences in the activation of drugs metabolized by these enzymes can- not be considered as a genetic component of human lon- gevity.

ture could in fact turn out to be centenarians, however, overlap between the two populations can be expected to be rather infrequent since the known fraction of centenarians in the French population is around 1 in 10 000.