Abstract
Methylglyoxal is a ketoaldehyde that reacts readily under physiological conditions with biologically relevant ligands, such as amine and sulfhydryl groups. It is produced in mammalian cells primarily as a by-product of glycolysis. The level of glucose, L-glutamine and fetal bovine serum in culture media was found to significantly affect levels of intracellular methylglyoxal in Chinese hamster ovary cells. Medium with 25 mM glucose and 5 mM L-glutamine caused an increase in free methylglyoxal levels of 90 to 100% relative to medium containing 5 mM glucose and 2 mM L-glutamine. Both of these media compositions are representative of those found in commercially available media. Pseudomonas putida glyoxalase I was expressed in Chinese hamster ovary cells to enhance methylglyoxal detoxification. The Chinese hamster ovary cell clones showed an 80 to 90% decrease in free methylglyoxal levels. The colony-forming ability of these cells was compared to wild-type Chinese hamster ovary cells under conditions found to cause elevated methylglyoxal levels. The wild-type cells showed a 10% decrease in colony-forming ability relative to the clones. This decrease was found to be statistically significant (P>0.99) by analysis of variance. The variation in colony-forming ability amongst the clones was statistically insignificant. More importantly, the clones shoed increased colony-forming ability relative to the wild-type cells under conditions of higher methylglyoxal production with fair to good statistical significance (P>0.75 to P>0.95). This result is the first quantifiable evidence that endogenously produced methylglyoxal can negatively affect cell function under conditions found in animal cell culture.
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Abbreviations
- ANOVA:
-
analysis of variance
- CHO:
-
Chinese hamster ovary cells
- CFA:
-
colony-forming ability
- dhfr :
-
gene for dihydrofolate reductase
- DHAP:
-
dihydroxyacetone phosphate
- FBS:
-
fetal bovine serum
- G-3-P:
-
glyceraldehyde-3-phosphate
- GloI:
-
glyoxalase I
- GloII:
-
glyoxalase II
- GSH:
-
reduced glutathione
- HPLC:
-
high-performance liquid chromatography
- IMDM:
-
Iscove's modified Dulbecco's medium
- MTX:
-
methotrexate
- 2-MQ:
-
2-methylquinoxaline
- 5-MQ:
-
5-methylquinoxaline
- MEM:
-
minimal essential medium
- Pi :
-
inorganic phosphate
- PCA:
-
perchloric acid
- o-PD:
-
o-phenylenediamine
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Chaplen, F.W.R., Fahl, W.E. & Cameron, D.C. Effect of endogenous methylglyoxal on Chinese hamster ovary cells grown in culture. Cytotechnology 22, 33–42 (1996). https://doi.org/10.1007/BF00353922
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DOI: https://doi.org/10.1007/BF00353922