Ammonia Affects Astroglial Proliferation in Culture

Abstract

Primary cultures of rat astroglial cells were exposed to 1, 3 and 5 mM NH4Cl for up to 10 days. Dose- and time-dependent reductions in cell numbers were seen, plus an increase in the proportion of cells in the S phase. The DNA content was reduced in the treated cells, and BrdU incorporation diminished. However, neither ammonia nor ammonia plus glutamine had any effect on DNA polymerase activity. iTRAQ analysis showed that exposure to ammonia induced a significant reduction in histone and heterochromatin protein 1 expression. A reduction in cell viability was also noted. The ammonia-induced reduction of proliferative activity in these cultured astroglial cells seems to be due to a delay in the completion of the S phase provoked by the inhibition of chromatin protein synthesis.

Fig 1. Effect of ammonia and serum on astroglial proliferation.

Effect of ammonia (NH₄Cl) exposure on astroglial proliferation was tested at two FBS concentrations (0.5 and 5%). **p<0.01. The percentage value is calculated taking the value for the 5% FBS control as 100% (1.65 x 10⁵ cells). Columns and bars represent means and SDs.

Fig 2. Effect of ammonia on astroglial proliferation.

Change in astroglial cell number after 10 days of exposure to ammonia. Standard deviations are not shown for 1 and 3 days since differences between the control and ammonia-treated cells were not significant at these times. *p<0.05, **p<0.01.

Fig 3. Effect of ammonia on viability of astroglial cells.

MTT viability assay of astroglial cells exposed to ammonia. Points and bars represent means and SDs. No SD values are included for 1, 3 and 5 days since they were <10% different to the mean.

Fig 4. Effect of ammonia on cell cycle phases.

Cell cycle analysis after 10 days of exposure to 1, 3 and 5 mM ammonia. The dotted lines are included to indicate the trends for the cell cycle phases. Columns and bars represent means and SDs. The percentages of cells in the phases considered (G₀-G₁, S and G₂-M) are included for every experimental condition; it should be noted that the sum of the three phases is 100% (10⁴ cells).

Fig 5. Effect of ammonia on DNA content.

(A) Four rows of the in-cell DNA content analysis plate; note that ammonia exposure reduced the fluorescence signal. The row shows only nine results since the external wells were not seeded and the control point without fluorescent dye is not included. (B) Plot showing the change in well DNA content after ammonia exposure. Points and bars represent means and SDs. **p<0.05, ***p<0.01.

Fig 6. Effect of ammonia on BrdU incorporation.

(A) BrdU incorporation by astroglial cells following exposure to ammonia. Points and bars represent means and SD. No significant changes were observed. (B) Cultured cells showing BrdU incorporation (red): (1) 1 day control, (2) 3 days after exposure to 5 mM ammonia. Scale bar: 10 μm.

Fig 7. Effect of ammonia on DNA polymerase.

Effect of ammonia (NH₄Cl) on the activity of DNA polymerase, analyzed via real-time PCR. Note that the Ct variation ranges between 20 and 21. Columns and bars represent means and SDs. No significant changes were observed.