The additional compounds tested at lower concentrations were chlorfenapyr, rotenone, triclocarban, and triethyltin bromide at 0.25, 0.1, and 0.05 [micro]M; and pinacyanol chloride, tested at 0.25, 0.1, 0.05, 0.05, 0.025, 0.01, and 0.005 [micro]M.
As shown in Excel Table S1, pinacyanol chloride was the most potent active compound, based on having the lowest [IC.sub.50] values in one of the two assays, with [IC.sub.50] of 0.04 and 0.02 [micro]M in rat primary hepatocytes and HepG2 cells, respectively, followed by chlorfenapyr (0.07 [micro]M in rat hepatocytes, 0.04 [micro]M in HepG2 cells), gramicidin (0.2 [micro]M in rat hepatocytes, 0.04 [micro]M in HepG2 cells), triclocarban (0.17 [micro]M in rat hepatocytes, 0.17 [micro]M in HepG2 cells), 2,6-di-tert-butyl-4-nitrophenol (0.29 [micro]M in rat hepatocytes, 0.11 [micro]M in HepG2 cells), niclosamide (0.44 [micro]M in rat hepatocytes, 0.10 [micro]M in HepG2 cells), and fluorosalan (0.42 [micro]M in rat hepatocytes, 0.19 [micro]M in HepG2 cells).
Of note, rotenone had a higher potency ([IC.sub.50] = 0.36 [micro]M) in rat primary hepatocytes than did hNSC ([IC.sub.50] = 23.7 [micro]M) and HepG2 cells ([IC.sub.50] = 44.7 [micro]M), whereas pinacyanol was more potent in hNSC ([IC.sub.50] = 0.04 [micro]M) and HepG2 cell ([IC.sub.50] = 0.04 [micro]M) than rat primary hepatocytes ([IC.sub.50] = 0.31 [micro]M).
Other compounds activated only the p53 pathway--including niclosamide, rafoxanide, and chlorfenapyr--or the Nrf2/ARE pathway--including pinacyanol, 1,8-dihydroxy-4, 5-dinitroanthraquinone, 2-aminoanthraquinone, 2-hydrazino-4-(4-aminophenyl) thiazole, 2-hydrazino-4-(4-nitrophenyl)thiazole, and 2-hydrazino-4-phenylthiazole (Table 2).
Picoxystrobin (Figure 3E,F) had effects similar to antimycin A, although pinacyanol chloride (Figure 3G,H) had slightly milder inhibitory effects on both glutamate/malate-driven respiration and succinate-driven respiration.
For example, rotenone and pinacyanol specifically inhibited complex I in a concentration-dependent manner (Figure 5A).
Moreover, pinacyanol chloride (2) is larger in size and would yield better information on dye polymer interaction.
Pinacyanol chloride (Pcyn) was obtained from Acros media and used as received.
The effect of alcohols namely, methanol, ethanol, 2-propanol and urea on the spectra of the pure dye was studied by measuring absorbance of the pure dye solution at 492 nm in case of acridine orange and at 600 nm in case of pinacyanol chloride in the presence of alcohols and urea.
The absorbances were measured at 492 nm and 457 nm in case of acridine orange and at 600 nm and 486 nm in case of pinacyanol chloride.
The absorption spectra of acridine orange (1) and pinacyanol chloride (2) at various concentrations are shown in figures 2 and 3, respectively.
While in case of pinacyanol chloride-heparin complex the stoichiometry is 1:2 and the binding is at alternate anionic sites.