ToxicologyLetters

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a b c a a c d Beate Pesch , Rainer Düsing , Sylvia Rabstein , Volker Harth , , c a d e Dagmar Grentrup , Thomas Brüning , Olfert Landt , ,∗ c c

Dedicated to the late Christian Hodel

Abstract

In order to investigate possible associations of genetic variants in genes of xenobiotic metabolism with longevity, we allele frequencies and genotype distributions of polymorphic genes between 205 octogenarians and a non-cancer reference 3801 T > C and CYP1B1 V432L, and in the glutathione S-transferase genes GSTM1 (deletion), GSTT1 (deletion), and (I105V). In octogenarians, the CYP1B1 432L allele was less prevalent than in the reference group (allele frequency 0.49 0.60; odds ratio, OR, 0.63, 95% confidence limits (CI) 0.40–1.00). Octogenarians turned out to have marginally significant GSTM1 negatives (frequency 0.56 versus 0.48; OR 1.41, 95% CI 0.97–2.05), but less GSTT1 deficient genotypes 0.14 versus 0.21; OR 0.64; 95% CI 0.38–1.06). 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 1.00–7.38). 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 to develop cancer in Ger- man male octogenarians is about 300 times the risk of adolescents, and for females 200 times,

0378-4274/$ – see front matter © 2004 Published by Elsevier Ireland Ltd. doi:10.1016/j.toxlet.2004.01.025

B. Pesch et al. / Toxicology Letters 151 (2004) 283–290

2.2. Genotypings

respectively (Schön et al., 1999). The exponential shape of the age-specific incidence of cancer sup- ports a stochastic process of accumulating errors at different levels of aging. Aging-related declines in toxic defence mechanisms have been related to gen- eration of reactive oxygen species (ROS; Beckman and Ames, 1998) and the decrease in detoxi- fying agents such as glutathione (van Lieshout and Peters, 1998). There is evidence that gene variants that affect metabolism of xenobiotics may modify the life span. The isoenzyme families cytochrome P450 (CYP) and glutathione S-transferases (GST) metabolize a wide range of substrates and are of importance for the balance of the oxidative status. Se- quence variations in these genes have been assumed to predispose to a variety of age-related diseases. We, therefore, conducted a study on 205 octogenari- ans (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 iso- lated 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 Hy- bridization Probes Kit (Roche, Mannheim, Germany). Both the PCR primers and the fluorescent-labeled detection probes were synthesized by TIB MOL- BIOL, 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 accord- ing 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 Corpo- ration, Cambridge, MA, USA). We investigated the impact of age, gender, and cancer on the allelic vari- ation. The risk estimates were not adjusted for these stratification factors.

2. Subjects and methods

2.1. Subjects

Two-hundred-and-five octogenarians were re- cruited from three different homes for the aged in the city of Bonn, Germany (Lottermoser et al., 2001). Octogenarians were eligible if aged 80 years or older and being able to follow an in- terview. As reference population, 294 unrelated non-cancer controls aged 17–79 years were ascer- tained from the outpatient clinics of the Univer- sity of Bonn and of the Environmental Health Re- search Institute in Düsseldorf, Germany (Ko et al., 2001). Blood samples were collected, with approval of the locally responsible Ethics Commission, and written informed consent. Body mass index (BMI) was obtained by dividing the weight in kilograms by height measured in meters, squared. Obesity was defined as BMI ≥ 30 kg/m2. Smoking was assessed as ever or never having smoked. Any diagnosis of can- cer, diabetes mellitus, or myocardial infarction was self-reported.

3. Results

Two-hundred-and-five German octogenarians aged 80–98 years were recruited from homes for the aged, representing a selected subgroup of the very old. We compared the prevalence of lifestyle factors and chronic diseases in these octogenarians with 374 West Germans aged 70–79 years from the German Health Survey 1998 (Bellach, 1999; see Table 1). Among the octogenarians, ever smoking and obesity were less prevalent than in Germans aged 70–79 years from the Health Survey (Bellach, 1999; see Table 1). The octogenarians did not represent a healthy group; 34 (16%) reported having suffered from cancer, 29 (14%) from diabetes mellitus, 37 (18%) from my- ocardial infarction, and 36 (18%) from stroke. The

B. Pesch et al. / Toxicology Letters 151 (2004) 283–290

Basic characteristics of the study population by age and gender