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. 2011 May 30;191(1-3):269-77.
doi: 10.1016/j.cbi.2011.02.016. Epub 2011 Feb 19.

"V体育官网" Aldehyde dehydrogenase 7A1 (ALDH7A1) attenuates reactive aldehyde and oxidative stress induced cytotoxicity

Chad Brocker  1 , Miriam Cantore, Paola Failli, Vasilis Vasiliou
Affiliations

Aldehyde dehydrogenase 7A1 (ALDH7A1) attenuates reactive aldehyde and oxidative stress induced cytotoxicity

Chad Brocker et al. Chem Biol Interact. .

Abstract

Mammalian aldehyde dehydrogenase 7A1 (ALDH7A1) is homologous to plant ALDH7B1 which protects against various forms of stress such as increased salinity, dehydration and treatment with oxidants or pesticides VSports手机版. Deleterious mutations in human ALDH7A1 are responsible for pyridoxine-dependent and folinic acid-responsive seizures. In previous studies, we have shown that human ALDH7A1 protects against hyperosmotic stress presumably through the generation of betaine, an important cellular osmolyte, formed from betaine aldehyde. Hyperosmotic stress is coupled to an increase in oxidative stress and lipid peroxidation (LPO). In this study, cell viability assays revealed that stable expression of mitochondrial ALDH7A1 in Chinese hamster ovary (CHO) cells provides significant protection against treatment with the LPO-derived aldehydes hexanal and 4-hydroxy-2-nonenal (4HNE) implicating a protective function for the enzyme during oxidative stress. A significant increase in cell survival was also observed in CHO cells expressing either mitochondrial or cytosolic ALDH7A1 treated with increasing concentrations of hydrogen peroxide (H(2)O(2)) or 4HNE, providing further evidence for anti-oxidant activity. In vitro enzyme activity assays indicate that human ALDH7A1 is sensitive to oxidation and that efficiency can be at least partially restored by incubating recombinant protein with the thiol reducing agent β-mercaptoethanol (BME). We also show that after reactivation with BME, recombinant ALDH7A1 is capable of metabolizing the reactive aldehyde 4HNE. In conclusion, ALDH7A1 mechanistically appears to provide cells protection through multiple pathways including the removal of toxic LPO-derived aldehydes in addition to osmolyte generation. .

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Conflict of interest statement

Conflict of interest

The authors declare no conflict of interest associated with this manuscript.

Figures

Fig. 1
Fig. 1
ALDH7A1 transgene expression in CHO cells. ALDH7A1 protein levels in both stably (A) and transiently (C) transfected cell lysates were analyzed by Western blot analysis. 20 μg cell lysates were ran per well. 30 ng recombinant human ALDH7A1 was included as a positive control (rALDH7A1). Densitometry was then used to compare protein levels between cell lines (B and D). ALDH7A1 protein levels were normalized to the respective β-actin expression. Density is displayed as the means ± standard deviation from three separate experiments (*p < 0.05).
Fig. 2
Fig. 2
Mitochondrial and cytosolic ALDH7A1 provide significant protection against hydrogen peroxide (H2O2)-induced cytotoxicity. CHO cells were transiently transfected with either mitochondrial (CHO-ALDH7A1v1) (A) or cytosolic (CHO-ALDH7A1v2) (B) ALDH7A1 expression constructs then treated with increasing concentrations of H2O2 (0–5 mM) for 16 h and cell survival accessed using SRB assays. Vector transfections were used as a negative control (CHO-Vector). Cell survival data was normalized in respect to untreated control (% untreated). Images of treated cells show significant protection at the indicated concentrations (C). Asterisks immediately above bars denote significant difference from vector control. Data represent means ± SD from triplicate assays (*p < 0.05; ***p < 0.001).
Fig. 3
Fig. 3
Mitochondrial and cytosolic ALDH7A1 provide significant protection against 4-hydroxy-2-nonenal (4HNE)-induced cytotoxicity. CHO cells were transiently transfected with either mitochondrial (CHO-ALDH7A1v1) (A) or cytosolic (CHO-ALDH7A1v2) (B) ALDH7A1 expression constructs then treated with increasing concentrations of 4HNE (0–100 μM) for 16 h and cell survival accessed using SRB assays vector transfections were used as a negative control (CHO-Vector). EtOH alone was used to determine the effect of this vehicle. Cell survival data was normalized in respect to untreated control (% untreated). Images of treated cells support significant protection at the indicated concentrations (C). Asterisks immediately above bars denote significant difference from vector control. Data represent means ± SD from triplicate assays (**p < 0.01; ***p < 0.001).
Fig. 4
Fig. 4
Mitochondrial ALDH7A1 expression provides significant protection against α-aminoadipic semialdehyde (AASA)-induced cytotoxicity. CHO cells stably transfected with either vector (CHO-Vector) or mitochondrial ALDH7A1 (CHO-ALDH7A1v1 #35) were treated in 96-well tissue culture plates with various concentrations of AASA (0–10 mM) for 24 h and assayed for mitochondrial function by MTT assay. Cell viability data was normalized in respect to untreated control (% untreated). Asterisks immediately above bars denote significant difference from vector control. Data are presented as a mean ± standard deviation from 27 different biological samples from three separate experiments (*p < 0.05).
Fig. 5
Fig. 5
ALDH7A1 activity is sensitive to oxidation. Incubation of ALDH7A1 with the reducing agent BME restores catalytic activity. In vitro activity assays with recombinant human protein revealed an approximately 4 fold increase in initial velocity (V0) at all propanal concentrations (0.25 mM, 0.75 mM & 1.0 mM) tested when compared to non-activated enzyme. Asterisks immediately above bars denote significant difference from vector control (***p < 0.001).
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