Are changes in olanzapine-induced liver enzyme levels associated with GSTT1, GSTM1, GSTP1, and OGG1 gene polymorphisms?

For genotyping and enzyme determination we collected venous blood (9 mL) in EDTA tubes for biochemical and DNA analyses before olanzapine treatment (T1, baseline), 10±3 days after starting olanzapine treatment (T2), and 3±1 months after treatment (T3), based on previously published studies (19, 20).

DNA was isolated from the whole blood using the sodium perchlorate/chloroform method described in detail earlier (21). For GSTM1 and GSTT1 genotyping we used the multiplex polymerase chain reaction (PCR) and for GSTP1 rs1695 (313 A>G, Ile105Val) and OGG1 rs1052133 (1245 C>G, Ser326Cys) restriction fragment length polymorphism (RFLP)-PCR as described earlier (22).

A partial gene deletion at the GSTM1 locus on chromosome 1p13.3 (GSTM1 null genotype) results in the complete absence of GSTM1 enzyme activity. To detect this deletion we used the following PCR primers: 5′-CGC CAT CTT GTG CTA CAT TGC CCG-3′ (primer 1), 5′-ATC TTC TCC TCT TCT GTC TC-3′ (primer 2), and 5′-TTC TGG ATT GTA GCA GAT CA-3′ (primer 3). PCR was performed in a total volume of 10 µL consisting of 100 ng of genomic DNA, 0.25 mmol/L dNTP, 1.5 mmol/L MgCl₂, 0.4 µmol/L of each primer, and 1 unit of Taq DNA polymerase in 1 × PCR buffer. The PCR program consisted of a 2 min initial denaturation step at 94 °C, followed by 35 cycles of 1 min denaturation at 94 °C, 90 s annealing at 53 °C, and 2 min elongation at 72 °C. The final elongation was 10 min at 72 °C. The specific size of the GSTM1 gene PCR product (231 bp) and a control band (158 bp) were assessed after electrophoresis on a 2 % agarose gel.

The GSTT1 gene deletion on chromosome 22q11.23 was determined with the PCR using the 5′-AGG CAG CAG TGG GGG AGG ACC-3′ forward primer and the 5′-CTC ACC GGA TCA TGG CCA GCA-3′ reverse primer. PCR was performed in a total volume of 10 µL consisting of 100 ng of genomic DNA, 0.2 mmol/L dNTP, 2 mmol/L MgCl₂, 0.25 µmol/L of each primer, and 1 unit of Taq DNA polymerase in 1 × PCR buffer. The PCR program consisted of a 5 min initial denaturation step at 94 °C, followed by 30 cycles of 1 min denaturation at 94 °C, 1 min annealing at 60 °C, and 2 min elongation at 72 °C. The final elongation was 10 min at 72 °C. The specific sizes of the GSTT1 product (138 bp) and a control band (158 bp) were assessed after electrophoresis on a 2 % agarose gel.

An A to G polymorphism at a nucleotide in the GSTP1 gene 313 results in an amino acid substitution (Ile105Val). This residue lies in the enzyme’s substrate-binding site, and the polymorphism has been shown to affect enzyme activity (22). To detect it we used the following primers: 5′-ACC CCA GGG CTC TAT GGG AA-3′ (forward) and 5′-TGA GGG CAC AAG AAG CCC CT-3′ (reverse). PCR was performed in a total volume of 20 µL consisting of 100 ng of genomic DNA, 0.2 mmol/L dNTP, 1.5 mmol/L MgCl₂, 0.3 µmol/L of each primer, and 1 unit of Taq DNA polymerase in 1 × PCR buffer. The PCR program consisted of a 5 min initial denaturation step at 94 °C, followed by 30 cycles of 30 s denaturation at 94 °C, 30 s annealing at 57 °C, and 30 s elongation at 72 °C. The final elongation was 7 min at 72 °C. After the PCR, a 10 µL aliquot of the amplification product was digested overnight at 37 °C with 4 units of BsmAI. The digestion products were separated after electrophoresis on a 3 % NuSieve agarose gel, and the DNA fragments were visualised with 10 mg/mL ethidium bromide. The specific sizes of digestion products were as follows: Ile/Ile (wild) 176 bp, Ile/Val (heterozygous) 176, 93, and 83 bp, and Val/Val (mutant) 93 and 83 bp.

To genotype for the OGG1 Ser326Cys we used the following primers: 5′-ACT GTC ACT AGT CTC ACC AG-3′ (OGG1-forward 5′) and 5′-TGA ATT CGG AAG GTG CTT GGG GAA T-3′ (OGG1-reverse 3′). PCR was performed in a total volume of 20 µL consisting of 100 ng of genomic DNA, 0.25 mmol/L dNTP, 1.5 mmol/L MgCl₂, 0.3 pmol of each primer, and 1 unit of Taq DNA polymerase in 1 × PCR buffer. The PCR program consisted of a 2 min initial denaturation step at 94 °C, followed by 33 cycles of 15 s denaturation at 94 °C, 30 s annealing at 60 °C, and 35 s elongation at 72 °C. The final elongation was 10 min at 72 °C. PCR yielded a 207 base pair product. After PCR, a 10 µL aliquot of the amplification product was digested overnight at 37 °C with 2 units Fnu4HI. The digestion products were separated after electrophoresis on a 3 % NuSieve agarose gel, and the DNA fragments were visualised with 10 mg/mL ethidium bromide. The specific sizes of digestion products were as follows: Ser/Ser (wild) 207 bp, Ser/Cys (heterozygous) 207, 107, and 100 bp, and Cys/Cys (mutant) 107 and 100 bp.

Serum olanzapine concentrations were measured with liquid chromatography–electrospray-ionisation-tandem mass spectrometry (LC-ESI-MS/MS) (Shimadzu LCMS-8030, Tokyo, Japan) following the validated method of Uřinovská et al. (23) with slight modifications as described earlier (24).

Serum levels of ALT and AST of patients were obtained from the hospital, whereas serum α-GST was measured with the enzyme-linked immunosorbent Assay (ELISA) according to the manufacturer’s instructions (Microplate Assay for GSTA Product Number: GS41, Oxford Biomedical Research Inc., Oxford, MI, USA).

First we ran the power analysis with the G*power tool, release 3.1 (Heinrich Heine University, Düsseldorf, Germany) (25), which yielded a range from 0.85 to 0.93, confirming that our sample size was sufficient for further analysis. The obtained data were further analysed using the IBM SPSS Statistics version 17.0 (IBM Corporation, Armonk, NY, USA). Genotype distribution was checked against the Hardy-Weinberg equilibrium. The normality of distribution was determined with the Shapiro-Wilk test and the homogeneity of variances with the Levene test. Where applicable, descriptive statistics for continuous variables were expressed as means ± standard deviations (SD) or medians and quartile 1–3 range. The number of cases and percentages were used for categorical data. Mean differences between groups were compared using the Student’s t-test or Mann-Whitney U test. Differences between time points were evaluated for significance with the repeated measures analysis of variance (ANOVA) using the Wilks’ lambda or Friedman test where applicable. If they were statistically significant, we then ran the Bonferroni correction to control for type I errors for all possible multiple comparisons. Degrees of association between continuous variables were evaluated with Spearman’s rank correlation. A p-value of <0.05 was considered statistically significant.

Table 3 shows that the first 10±3 days of treatment with olanzapine increased the levels of all enzymes compared to their baseline values (T2 vs T1). However, only ALT rose significantly at T2 compared to T1 and dropped to baseline levels at T3, whereas the other two enzymes did not change significantly.

Serum ALT was above the upper limit of normal (ULN of <40 U/L) in seven patients at T2 and in one patient at T3. Only one patient had ALT>2xULN at T2 (clinically mild elevation).

Serum AST levels were above ULN of <40 U/L in six patients at T2 and in one patient at T3. All elevations were below <2×ULN.

Tables 4–6 show no significant associations between gene polymorphisms and the levels of liver enzymes measured at the three time points, save for the association between the GSTP1 Ile/Val + Val/Val polymorphism and ALT (p=0.014).