I
Index
Abstract III
1. Introduction 1
1.1 Ethanol toxicokinetics and metabolism
1
1.2 Alcohol dehydrogenases
4
1.3 Aldehyde dehydrogenases
7
1.4 ADH and ALDH transcriptional activity
9
1.5 Microsomal CYP2E1
11
1.6 Glutathione S-transferases
12
1.7 Studies of alcohol elimination rate and physiological factors governing alcohol disposition
14
1.8 Alcohol impact on basic social abilities and behavior
16
2. Aim of the study 20
3. Materials and Methods 21
3.1 Selection of SNPs
21
II
3.2.1 Calculation of the administered dose of wine
23
3.2.2 DNA extraction
24
3.2.3 Genotyping
25
3.2.4 Comparison between genotype and phenotype
25
3.3 Effect of rs886205 on ALDH2 ethanol-induced gene expression
26
3.3.1 Drinking experiments
26
3.3.2 RNA purification
27
3.3.3 cDNA synthesis
28
3.3.4 RT-qPCR
28
3.4 Effect of alcohol consumption on driving skills
30
3.4.1 DAT1 genotyping
30
4. Results 31
4.1 Interindividual variability in ethanol elimination rate
31
4.2 Effect of rs886205 on ALDH2 ethanol-induced gene expression
37
4.3 Effect of alcohol consumption on driving skills
38
5. Discussion 45
III
Abstract
The large interindividual and interethnic variability in the response to alcohol consumption comes from a combination of genetic and environmental factors, which affect ethanol toxicokinetics. About 90% of ethanol is metabolized in the liver, where the alcohol dehydrogenase (ADH) enzymes oxidize ethanol to acetaldehyde, and the aldehyde dehydrogenase (ALDH) enzymes convert acetaldehyde to acetate. Only 10% of ethanol is metabolized by the microsomal CYP2E1. All these enzymes have been shown to be polymorphic, giving rise to altered phenotypes: fast or slow metabolizers of ethanol. Electronic breathalyzer tests were organized to investigate whether exists a variation of phenotype (slow or fast metabolizers of ethanol) corresponding to different genotypes for a spectrum of single nucleotide polymorphisms (SNPs) residing in the genes involved in ethanol toxico-kinetics. After ethanol ingestion, variation of BAC was monitored over time in order to draw the corresponding disposal curve. The relationship between genotype and phenotype was investigated calculating the Area Under the Curve (AUC) for each individual. The analyses showed a significant association between the AUC values and the genotype, only for the allelic variant rs1614972 within the intron 8 of the ADH1C gene and revealed an influence of the time of day when the test was conducted (morning or afternoon) on ethanol elimination rate.
Moreover, measuring the time of reaction to a sudden slip of the vehicle during a simulated driving test, we studied the effect of alcohol consumption on driving skills, and we further investigated the possible role of the genes of ethanol metabolism and of the DAT1 gene in the individual physiological response. DAT1 is considered the primary candidate gene for the attention deficit hyperactivity disorder (ADHD) and contains a 40 bp variable number tandem repeat (VNTR) in its 3’-UTR region. With regard to polymorphisms in the metabolic genes, only rs11936869 and rs1693482, both residing in the ADH1C gene,
IV
appeared to affect the reaction times in the driving simulation. Concerning the DAT1 gene and its involvement in the decrement of driving performance, our analysis reveals the association of the 10 repeat allele with an increased time of reaction in simulated driving, and a protective role of the homozygous 9 repeat genotype.
