Phenolsulfonphthalein (phenol red) metabolism in primary monolayer cultures of adult rat hepatocytes

Abstract

The sulfonic acid dye, phenolsulfonphthalein (PSP or phenol red), has been incorporated as a pH indicator in many tissue culture media formulations since the emergence of tissue culture methodologies. The present study was designed to examine the pathway, time course, and degree of metabolism of this anionic dye in monolayer cultures of adult rat hepatocytes. Thin layer chromatographic studies coupled with beta-glucuronidase studies show that glucuronidation is the major metabolic pathway for PSP in vitro. About 20% of the dye is metabolized in the first 24 h, but this functional activity is decreased by approximately half at 48 h, and even further at 72 h of culture. This metabolic activity was not affected by continuous exposure to the dye. The effect of PSP concentration on its rate of metabolism by the adult rat hepatocyte in culture seemed to be biphasic, and at concentrations of less than 100 microM there was indication of a saturable process. Although PSP seemed not to be toxic to hepatocyte cultures, it is partially metabolized by these cells (as opposed to no observed metabolism in human fibroblasts or HeLa cells). Therefore, its incorporation into tissue culture media formulations for use in hepatocyte cultures should be avoided, especially when studying the mechanism(s) of glucuronidation or metabolic pathways thought to be affected by this anionic dye.

Circadian Clock Is Involved in Regulation of Hepatobiliary Transport Mediated by Multidrug Resistance-Associated Protein 2

“…Considering that PSP is converted to a glucuronide conjugate in the liver, these data suggested that the generation of PSP metabolites might decrease, while the biliary excretion via Mrp2 increased in WT-ZT12 and PDK-ZT00. 39 Although we could not measure glucuronide metabolite of PSP, the mRNA expression of uridine 5'-diphospho-glucuronosyltransferase (Ugt) showed a different circadian rhythm, depending on the subtypes in WT mouse liver as shown in Figure 5 and the previous report. 16 Moreover, in the PDK mouse, the mRNA expression of Ugt1a1 and Ugt1a6 slightly decreased by 0.77-and 0.70-fold, respectively, while that of Ugt1a2 significantly increased by 5.38-fold compared to WT mice at ZT00 (Figs.…”

Technique for the Kinetic Characterization of the Metabolic Reactions of Hepatocytes in Adhesion Culture

“…Figure 8 shows that PER increased linearly with increasing PSP concentrations in the medium. Also Driscoll et al (10) reported PERs linearly increasing with PSP concentration, up to 250 μM PSP: above that, they report a less‐than‐linear PER dependence on PSP concentration. Such a discrepancy is possibly to be blamed on the fact that, in their case, PER was estimated after only 3 h from cell isolation and cells might have not yet completed their adaptation to the substratum (28); cell activity losses might have also contributed to the different PERs.…”

“…Urea concentration was assayed by the enzymatic urease method (Sigma, St. Louis, MO). PSP concentration was measured colorimetrically: after centrifugation, 0.1 mL of the supernatant was added to 2 mL of pH 10, 0.5 M glycine−NaOH buffer and the absorbance of the solution was read at 546 nm (10).…”

“…The technique was used to characterize the dependence on ammonia and PSP concentration of the rate of ammonia elimination (AER), urea synthesis (USR), and PSP elimination (PER), respectively. The latter reaction was chosen as a marker for hepatocyte ability to conjugate bilirubin (10). We characterized the dependence of AER and USR on ammonia concentrations ranging from 0 to 2 mM by supplementing the medium with suitable amounts of ammonium chloride.…”

Synergistic anticancer effect of plasma-activated infusion and salinomycin by targeting autophagy and mitochondrial morphology

“…However, conventional PAM includes many media components, such as the pH indicator phenol red. This compound undergoes specific metabolism to form a toxic substance in the liver (Driscoll et al, 1982). Thus, PAM may damage the liver in patients.…”