Figure 1 depicts the effects of various concentrations of butyrate (0.01–1.0 µM) against both Fe- and Mn-induced toxicities in SH-SY5Y cells. For both metals, we used a concentration of 60 µM because at this concentration significant toxicity (about 40%) is observed during the 24 h exposure. As seen, there was a concentration-dependent protection by butyrate against both iron [F(4,20) = 11.4, p < 0.01] and manganese [F(4,20) = 12.3, p < 0.0] toxicities with full protection at 1.0 µM butyrate. Butyrate by itself, at any concentration, did not affect the cell viability (data not shown)
Effects of various concentrations of butyrate (BUT) against iron (Fe)- and manganese (Mn)-induced toxicities. Cells were treated with Fe, Mn with and without BUT for 24 h and cell viability was determined by MTT. BUT was added 1 h before Fe or Mn. Values are mean ± SEM. **p < 0.01 compared to control. †p < 0.05, ††p < 0.01 compared to Fe or Mn only. N = 5 per treatment.
Figure 2 depicts the effect of various concentrations of nicotine (NIC) (0.01–1.0 µM) against Fe- and Mn-induced toxicities. Here also, there was a concentration-dependent protection by Nic [F(4,20) = 10.4, p < 0.01], similar to what we had observed previously (Getachew et al. 2019). This experiment was carried out to determine the concentrations of nicotine in these batch of cells to allow combination studies with butyrate
Effects of various concentrations of nicotine (NIC) against iron (Fe)- and manganese (Mn)-induced toxicities. Cells were treated with Fe, Mn with and without NIC for 24 h and cell viability was determined by MTT. NIC was added 1 h before Fe or Mn. Values are mean ± SEM. **p < 0.01 compared to control. †p < 0.05, ††p < 0.01 compared to Fe or Mn only. N = 5 per treatment.
Figure 3 depicts the effect of combination of an ineffective concentration of butyrate (BUT) with 2 ineffective concentrations of nicotine (NIC) against iron (Fe)-induced toxicity. As seen, the combination of the lowest NIC concentration with the ineffective concentration of BUT resulted in substantial protection (approximately 75%. P < 0.05) against Fe toxicity. The combination of higher ineffective concentration of NIC with the same concentration of BUT resulted in 100% protection (p < 0.01). Hence, a synergistic protection by the combination of ineffective concentrations of NIC and BUT against Fe-induced toxicity can be suggested.
Effects of combination of nicotine (NIC) and butyrate (BUT) against iron (Fe)-induced toxicity. Cells were treated with 2 ineffective concentrations of NIC and one ineffective concentration of BUT for 24 h. For each combination, NIC and BUT were added together 1 h before Fe. Cell viability was determined by MTT. Values are mean ± SEM. *p < 0.05, **p < 0.01 compared to control. †p < 0.05, ††p < 0.01 compared to Fe only. N = 5 per treatment.
Figure 4 depicts the effect of combination of ineffective concentration of butyrate (BUT) with 2 ineffective concentrations of nicotine (NIC) against manganese (Mn)-induced toxicity. As seen, the combination of the lowest NIC concentration with the ineffective concentration of BUT resulted in substantial protection (approximately 76%. P < 0.05) against Mn toxicity. The combination of higher ineffective concentration of NIC with the same concentration of BUT resulted in 100% protection (p < 0.01). Hence, a synergistic protection by the combination of ineffective concentrations of NIC and BUT against Mn-induced toxicity can be suggested.
Effects of combination of nicotine (Nic) and butyrate (BUT) against manganese (Mn)-induced toxicity. Cells were treated with 2 ineffective concentrations of Nic and one ineffective concentration of BUT for 24 h. For each combination, Nic and BUT were added together 1 h before Mn. Cell viability was determined by MTT. Values are mean ± SEM. *p < 0.05, **p < 0.01 compared to control. †p < 0.05, ††p < 0.01 compared to Mn only. N = 5 per treatment.
Figure 5 depicts the effect of mecamylamine (MEC), a non-selective nicotinic receptor antagonist on protective effects of nicotine (NIC) or butyrate (BUT) against iron (Fe)- or manganese (Mn)-induced toxicities. As seen, MEC (1.0 µM) completely blocked the protective effects of NIC against both Fe- and Mn- induced toxicities (p < 0.01). On the other hand, MEC did not affect the protective effect of BUT against either Fe- or Mn-induced toxicities, suggesting selective action of NIC via nicotinic receptor.
Effect of mecamylamine (MEC) on nicotine (NIC) or butyrate (BUT) protection against iron (Fe) or manganese (Mn) toxicities. MEC was added 1 h before NIC or BUT, which in turn, were added 1 h prior to Fe or Mn. Cell viability was assessed using MTT assay 24 h later. Values are mean ± SEM. **p < 0.01 compared to control, ††p < 0.01 compared to Fe or Mn only. N = 5 per treatment
Figure 6 depicts the effect of beta-hydroxy butyrate (BHB), a selective fatty acid 3 receptor (FA3R) antagonist, on protective effects of nicotine (NIC) or butyrate (BUT) against iron (Fe)- or manganese (Mn)-induced toxicities. As seen, BHB (10 µM) completely blocked the protective effects of BUT against both Fe and Mn (p < 0.01). On the other hand, BHB did not affect the protective effect of Nic against either Fe or Mn, suggesting selective action of BUT through FA3R.
BHB (10 µM) by itself did not have any effect on cell viability (data not shown).
Effect of beta-hydroxy butyrate (BHB), a selective fatty acid 3 receptor (FA3R) antagonist on nicotine (NIC) or butyrate (BUT) protection against iron (Fe)- or manganese (Mn)-induced toxicities. BHB was added 1 h before Nic or BUT, which in turn, were added 1 h prior to Fe or Mn. Cell viability was assessed using MTT assay 24 h later. Values are mean ± SEM. **p < 0.01 compared to control, ††p < 0.01 compared to Fe or Mn only. N = 5 per treatment.