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Eliciting the low-activity aldehyde dehydrogenase Asian phenotype by an antisense mechanism results in an aversion to ethanol.

Garver E - J. Exp. Med. (2001)

Bottom Line: Rat hepatoma cells incubated for 24 h with an antisense oligonucleotide (ASO-9) showed reductions in ALDH2 mRNA levels of 85% and ALDH2 (half-life of 22 h) activity of 55% equivalent to a >90% inhibition in ALDH2 synthesis.Base mismatches in the oligonucleotide rendered ASO-9 virtually inactive, confirming an antisense effect.Administration of ASO-9 (20 mg/kg/day for 4 d) to rats resulted in a 50% reduction in liver ALDH2 mRNA, a 40% inhibition in ALDH2 activity, and a fourfold (P < 0.001) increase in circulating plasma acetaldehyde levels after ethanol (1 g/kg) administration.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Anatomy and Cell Biology, and the Alcohol Research Center, Thomas Jefferson University, Philadelphia PA 19107, USA. emg002@jefferson.edu

ABSTRACT
A mutation in the gene encoding for the liver mitochondrial aldehyde dehydrogenase (ALDH2-2), present in some Asian populations, lowers or abolishes the activity of this enzyme and results in elevations in blood acetaldehyde upon ethanol consumption, a phenotype that greatly protects against alcohol abuse and alcoholism. We have determined whether the administration of antisense phosphorothioate oligonucleotides (ASOs) can mimic the low-activity ALDH2-2 Asian phenotype. Rat hepatoma cells incubated for 24 h with an antisense oligonucleotide (ASO-9) showed reductions in ALDH2 mRNA levels of 85% and ALDH2 (half-life of 22 h) activity of 55% equivalent to a >90% inhibition in ALDH2 synthesis. Glutamate dehydrogenase mRNA and activity remained unchanged. Base mismatches in the oligonucleotide rendered ASO-9 virtually inactive, confirming an antisense effect. Administration of ASO-9 (20 mg/kg/day for 4 d) to rats resulted in a 50% reduction in liver ALDH2 mRNA, a 40% inhibition in ALDH2 activity, and a fourfold (P < 0.001) increase in circulating plasma acetaldehyde levels after ethanol (1 g/kg) administration. Administration of ASO-9 to rats by osmotic pumps led to an aversion (-61%, P < 0.02) to ethanol. These studies provide a proof of principle that specific inhibition of gene expression can be used to mimic the protective effects afforded by the ALDH2-2 phenotype.

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Quantitation of ALDH2–1 and GDH steady-state mRNA by competitive RT-PCR. A1 and A2 show ALDH2 mRNA expression and B1 and B2 show GDH mRNA expression. H4-II-E-C3 hepatoma cells were incubated with Lipofectamine Plus™ in the presence of 1 μM ASO-9 (A1 and B1) or the 4-base mismatch of ASO-9 (A2 and B2, nucleotide sequence in Table ). The concentration shown represents the pg of ALDH2 mRNA competitor added/μg total RNA (A1 and A2) and ng GDH mRNA competitor added/μg total RNA (B1 and B2). As can be observed, lesser competitor was necessary to compete with ALDH2 mRNA in ASO-9–treated cells than in cells treated with the 4-base mismatch of ASO-9 (control). In three replicates, the relative concentration of ALDH mRNA for 4-base mismatch control cells was (indicated by arrows) 21.8 ± 8.3 pg/mg total RNA; that for the ASO-9–treated cells was 5.6 ± 0.5 pg/mg total RNA. ASO-9 did not affect the GDH mRNA levels when compared with the 4-base mismatch oligonucleotide. In three replicates, the relative concentration of GDH mRNA for 4-base mismatch control cells was (indicated by arrows) 9.65 ± 0.19 ng/mg total RNA; that for the ASO-9–treated cells was 9.26 ± 0.18 ng/mg total RNA.
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Figure 1: Quantitation of ALDH2–1 and GDH steady-state mRNA by competitive RT-PCR. A1 and A2 show ALDH2 mRNA expression and B1 and B2 show GDH mRNA expression. H4-II-E-C3 hepatoma cells were incubated with Lipofectamine Plus™ in the presence of 1 μM ASO-9 (A1 and B1) or the 4-base mismatch of ASO-9 (A2 and B2, nucleotide sequence in Table ). The concentration shown represents the pg of ALDH2 mRNA competitor added/μg total RNA (A1 and A2) and ng GDH mRNA competitor added/μg total RNA (B1 and B2). As can be observed, lesser competitor was necessary to compete with ALDH2 mRNA in ASO-9–treated cells than in cells treated with the 4-base mismatch of ASO-9 (control). In three replicates, the relative concentration of ALDH mRNA for 4-base mismatch control cells was (indicated by arrows) 21.8 ± 8.3 pg/mg total RNA; that for the ASO-9–treated cells was 5.6 ± 0.5 pg/mg total RNA. ASO-9 did not affect the GDH mRNA levels when compared with the 4-base mismatch oligonucleotide. In three replicates, the relative concentration of GDH mRNA for 4-base mismatch control cells was (indicated by arrows) 9.65 ± 0.19 ng/mg total RNA; that for the ASO-9–treated cells was 9.26 ± 0.18 ng/mg total RNA.

Mentions: The specificity of ASO-9 was also demonstrated by determining mRNA levels of ALDH2 and of GDH, another mitochondrial enzyme, in the same cells. Fig. 1 shows the reduction in mRNA ALDH2 afforded by ASO-9 (see Fig. 1aFig. 1) and its ineffectiveness on GDH mRNA levels (Fig. 1, B1).


Eliciting the low-activity aldehyde dehydrogenase Asian phenotype by an antisense mechanism results in an aversion to ethanol.

Garver E - J. Exp. Med. (2001)

Quantitation of ALDH2–1 and GDH steady-state mRNA by competitive RT-PCR. A1 and A2 show ALDH2 mRNA expression and B1 and B2 show GDH mRNA expression. H4-II-E-C3 hepatoma cells were incubated with Lipofectamine Plus™ in the presence of 1 μM ASO-9 (A1 and B1) or the 4-base mismatch of ASO-9 (A2 and B2, nucleotide sequence in Table ). The concentration shown represents the pg of ALDH2 mRNA competitor added/μg total RNA (A1 and A2) and ng GDH mRNA competitor added/μg total RNA (B1 and B2). As can be observed, lesser competitor was necessary to compete with ALDH2 mRNA in ASO-9–treated cells than in cells treated with the 4-base mismatch of ASO-9 (control). In three replicates, the relative concentration of ALDH mRNA for 4-base mismatch control cells was (indicated by arrows) 21.8 ± 8.3 pg/mg total RNA; that for the ASO-9–treated cells was 5.6 ± 0.5 pg/mg total RNA. ASO-9 did not affect the GDH mRNA levels when compared with the 4-base mismatch oligonucleotide. In three replicates, the relative concentration of GDH mRNA for 4-base mismatch control cells was (indicated by arrows) 9.65 ± 0.19 ng/mg total RNA; that for the ASO-9–treated cells was 9.26 ± 0.18 ng/mg total RNA.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2195938&req=5

Figure 1: Quantitation of ALDH2–1 and GDH steady-state mRNA by competitive RT-PCR. A1 and A2 show ALDH2 mRNA expression and B1 and B2 show GDH mRNA expression. H4-II-E-C3 hepatoma cells were incubated with Lipofectamine Plus™ in the presence of 1 μM ASO-9 (A1 and B1) or the 4-base mismatch of ASO-9 (A2 and B2, nucleotide sequence in Table ). The concentration shown represents the pg of ALDH2 mRNA competitor added/μg total RNA (A1 and A2) and ng GDH mRNA competitor added/μg total RNA (B1 and B2). As can be observed, lesser competitor was necessary to compete with ALDH2 mRNA in ASO-9–treated cells than in cells treated with the 4-base mismatch of ASO-9 (control). In three replicates, the relative concentration of ALDH mRNA for 4-base mismatch control cells was (indicated by arrows) 21.8 ± 8.3 pg/mg total RNA; that for the ASO-9–treated cells was 5.6 ± 0.5 pg/mg total RNA. ASO-9 did not affect the GDH mRNA levels when compared with the 4-base mismatch oligonucleotide. In three replicates, the relative concentration of GDH mRNA for 4-base mismatch control cells was (indicated by arrows) 9.65 ± 0.19 ng/mg total RNA; that for the ASO-9–treated cells was 9.26 ± 0.18 ng/mg total RNA.
Mentions: The specificity of ASO-9 was also demonstrated by determining mRNA levels of ALDH2 and of GDH, another mitochondrial enzyme, in the same cells. Fig. 1 shows the reduction in mRNA ALDH2 afforded by ASO-9 (see Fig. 1aFig. 1) and its ineffectiveness on GDH mRNA levels (Fig. 1, B1).

Bottom Line: Rat hepatoma cells incubated for 24 h with an antisense oligonucleotide (ASO-9) showed reductions in ALDH2 mRNA levels of 85% and ALDH2 (half-life of 22 h) activity of 55% equivalent to a >90% inhibition in ALDH2 synthesis.Base mismatches in the oligonucleotide rendered ASO-9 virtually inactive, confirming an antisense effect.Administration of ASO-9 (20 mg/kg/day for 4 d) to rats resulted in a 50% reduction in liver ALDH2 mRNA, a 40% inhibition in ALDH2 activity, and a fourfold (P < 0.001) increase in circulating plasma acetaldehyde levels after ethanol (1 g/kg) administration.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Anatomy and Cell Biology, and the Alcohol Research Center, Thomas Jefferson University, Philadelphia PA 19107, USA. emg002@jefferson.edu

ABSTRACT
A mutation in the gene encoding for the liver mitochondrial aldehyde dehydrogenase (ALDH2-2), present in some Asian populations, lowers or abolishes the activity of this enzyme and results in elevations in blood acetaldehyde upon ethanol consumption, a phenotype that greatly protects against alcohol abuse and alcoholism. We have determined whether the administration of antisense phosphorothioate oligonucleotides (ASOs) can mimic the low-activity ALDH2-2 Asian phenotype. Rat hepatoma cells incubated for 24 h with an antisense oligonucleotide (ASO-9) showed reductions in ALDH2 mRNA levels of 85% and ALDH2 (half-life of 22 h) activity of 55% equivalent to a >90% inhibition in ALDH2 synthesis. Glutamate dehydrogenase mRNA and activity remained unchanged. Base mismatches in the oligonucleotide rendered ASO-9 virtually inactive, confirming an antisense effect. Administration of ASO-9 (20 mg/kg/day for 4 d) to rats resulted in a 50% reduction in liver ALDH2 mRNA, a 40% inhibition in ALDH2 activity, and a fourfold (P < 0.001) increase in circulating plasma acetaldehyde levels after ethanol (1 g/kg) administration. Administration of ASO-9 to rats by osmotic pumps led to an aversion (-61%, P < 0.02) to ethanol. These studies provide a proof of principle that specific inhibition of gene expression can be used to mimic the protective effects afforded by the ALDH2-2 phenotype.

Show MeSH
Related in: MedlinePlus