Toxicology Letters www.elsevier.com/locate/toxlet

Beate Pesch, Rainer Dsing, Sylvia Rabstein, Volker Harth, Dagmar Grentrup, Thomas Brning, Olfert Landt

Dedicated to the late Christian Hodel

Abstract

In order to investigate possible associations of genetic variants in genes of xenobiotic metabolism with longevity, we studied allele frequencies and genotype distributions of polymorphic genes between 205 octogenarians and a non-cancer reference group. We analyzed CYP1A1 T461N, CYP1A1 3801 T > C and CYP1B1 V432L, and in the glutathione S-transferase genes GSTM1 (deletion), GSTT1 (deletion), and GSTP1 I105V. In octogenarians, the CYP1B1 432L allele was less prevalent than in the reference group (allele frequency 0.49 vs. 0.60; odds ratio OR 0.63, 95% confidence limits CI 0.401.00). Octogenarians turned out to have marginally significant GSTM1 negatives (frequency 0.56 versus 0.48; OR 1.41, 95% CI 0.972.05), but fewer GSTT1 deficient genotypes (frequency 0.14 versus 0.21; OR 0.64; 95% CI 0.381.06). In octogenarians without cancer, GSTT1 negative carriers were less than in the aged with cancer (frequency 0.12 versus 0.27; OR 2.81; 95% CI 1.007.38). Polymorphic metabolic genes could become relevant for processes of aging when toxic defense mechanisms decline.

Metabolism; Single nucleotide polymorphism; Aging; Cytochrome P-450; Glutathione S-transferase;

Age is a pronounced risk factor for most chronic human diseases. The risk of developing cancer in German male octogenarians is about 300 times the risk of adolescents, and for females 200 times (Schn et al., 1999). The exponential shape of the age-specific incidence of cancer supports a stochastic process of accumulating errors at different levels of aging. Aging-related declines in toxic defense mechanisms have been related to generation of reactive oxygen species (ROS; Beckman and Ames, 1998) and the decrease in detoxifying agents such as glutathione (van Lieshout and Peters, 1998). There is evidence that gene variants that affect metabolism of xenobiotics may modify the lifespan. The isoenzyme families cytochrome P450 (CYP) and glutathione S-transferases (GST) metabolize a wide range of substrates and are important for the balance of the oxidative status. Sequence variations in these genes have been assumed to predispose to a variety of age-related diseases. We conducted a study on 205 octogenarians (aged 80-98 years) and 294 non-cancer controls (aged 17-79 years) to investigate the association of gene deletions in GSTM1 and GSTT1 and of the sequence variations CYP1A1 3801 T > C, CYP1A1 T461N, CYP1B1 V432L, and GSTP1 I105V with aging.

From the study subjects, genomic DNA was isolated from whole blood using the QiAmp DNA Blood Maxi Kit (Qiagen, Hilden, Germany). PCR was performed on a LightCyclerTM instrument (Roche, Mannheim, Germany) using hybridization probes in combination with LightCyclerTM DNA Master Hybridization Probes Kit (Roche, Mannheim, Germany). Both the PCR primers and the fluorescent-labeled detection probes were synthesized by TIB MOLBIOL, Berlin, Germany. The PCR conditions, PCR primers, and fluorescent-labeled detection probes for genotyping CYP1A1, CYP1B1, GSTM1, GSTT1, and GSTP1 have been previously published (Ko et al., 2001). The reference sequences were defined according to the U.S. National Center for Biotechnology Information reference sequences (Pruitt and Maglott, 2001). Therefore, the variant allele is not always the minor allele. Allele frequencies, deviation from Hardy-Weinberg equilibrium (HWE), and OR with 95% CI for allele positivity (variant allele carriers versus homozygous reference genotype carriers) were calculated with a program of T.M. Strom and T.F. Wienker (URL: http://ihg.gsf.de/cgi-bin/hw/hwa1.pl) and StatXact, version 4 (CYTEL Software Corporation, Cambridge, MA, USA). We investigated the46.3%, 107 (36.4%), 51 (25.0%), 105 (51.5%), 48 (23.5%), 0.63, 0.40, 1.00, 0.60, 0.49, and 0.54 are percentages and ratios that are likely part of a table or chart and may not require editing.

For the following section, some abbreviations and technical terms may be difficult to understand without further context or knowledge of the subject matter.

---

In a German study on the possible role of genetic polymorphisms of three enzymes - GSTM1, N-acetyltransferase 2, and debrisoquine/sparteine hydrolase (CYP2D6) - in aging, there was no association with longevity (Muiras et al., 1998). By contrast, in an Italian study of polymorphisms of metabolic enzymes in healthy nonagenarians and centenarians, GSTT1 deletions were significantly more prevalent than in the younger reference group (28% versus 19%), and no association with longevity could be demonstrated for the GSTM1 deletion and for CYP1A1 polymorphisms (Taioli et al., 2001).

With respect to genetic variants investigated in Germans older or younger than 80 years, so far we could not demonstrate striking differences, and the differences found have to be considered carefully with respect to random variation because of multiple comparisons. These results confirm that metabolic susceptibility genes have not been found as strong risk modifiers for aging and age-related diseases as expected in the 1990s (Garte, 2001). A comprehensive database of the allelic variation in common metabolic genes has been established in the International Project on Genetic Susceptibility (GSEC) containing information on over 15,000 non-cancer controls (Garte et al., 2001). Facing the large variation in the human genome, one cannot expect that a single common gene variant may play a decisive role in either disease development or aging. Only a few rare sequence variants were found strongly associated with a disease risk, such as BRCA1 with breast cancer and genes resulting in the Werner syndrome with aging.

---