Caffeic acid-, chlorogenic acid-, and dihydrocaffeic acid-induced cytotoxicity involves metabolic oxidation
| Treatment | Cytotoxicity (% Trypan Blue Uptake) | % Hepatocyte GSH | |||
|---|---|---|---|---|---|
| 60 min | 120 min | 180 min | 30 min | 90 min | |
| Hepatocytes | 19 ± 2 | 21 ± 2 | 22 ± 1 | 100 | 100 |
| + caffeic acid (5 mM) | 22 ± 1 | 32 ± 3 | 41 ± 45-150 | 83 ± 3 | 71 ± 4 |
| + benzylimidazole (100 μM)5-b | 22 ± 2 | 27 ± 3 | 28 ± 3 | 94 ± 4 | 93 ± 6 |
| + dicumarol (20 μM)5-b | 28 ± 3 | 42 ± 4 | 56 ± 45-150 | 72 ± 4 | 40 ± 5 |
| +NaN3 (4 mM)5-b | 30 ± 3 | 49 ± 5 | 86 ± 65-150 | 51 ± 3 | 4 ± 3 |
| + GSH-depleted hepatocytes | 45 ± 4 | 48 ± 5 | 77 ± 65-150 | 9 ± 5 | 4 ± 3 |
| +4-propylcatechol (0.5 mM) | 33 ± 3 | 50 ± 2 | 76 ± 3 | 52 ± 3 | 39 ± 3 |
| + dicumarol (20 μM)5-b | 32 ± 2 | 92 ± 3 | 1005-150 | 26 ± 3 | 13 ± 2 |
| + dihydrocaffeic acid (5 mM) | 22 ± 3 | 23 ± 3 | 22 ± 2 | 100 | 100 |
| +dicumarol (20 μM)5-b | 25 ± 4 | 60 ± 4 | 85 ± 65-150 | 94 ± 6 | 64 ± 5 |
| + chlorogenic acid (5 mM) | 29 ± 2 | 28 ± 3 | 32 ± 3 | 66 ± 5 | 20 ± 3 |
| + dicumarol (20 μM)5-b | 33 ± 3 | 37 ± 4 | 42 ± 45-150 | 54 ± 3 | 5 ± 4 |
| +caffeic acid (1 mM) | 21 ± 2 | 22 ± 3 | 24 ± 2 | ||
| +H2O2 (10 mM glucose/1 unit/ml g.o)5-a 5-b | 33 ± 4 | 58 ± 5 | 78 ± 85-150 | ||
| + H2O2 +benzylimidazole (100 μM) | 24 ± 3 | 30 ± 2 | 32 ± 3 | ||
| + cumene hydroperoxide (130 μM)5-b | 41 ± 5 | 71 ± 6 | 92 ± 85-150 | ||
| + cumene hydroperoxide +benzylimidazole (100 μM) | 26 ± 3 | 32 ± 3 | 35 ± 4 | ||
| + tyrosinase (100 units/ml)5-b | 38 ± 4 | 62 ± 6 | 86 ± 75-150 | ||
Hepatocytes (10 ml) (106 cells/ml) were preincubated at 37°C in Krebs-Henseleit buffer, pH 7.4, with CA and benzylimidazole or hydrogen peroxide (glucose/glucose oxidase system) or cumene hydroperoxide or dicumarol or tyrosinase or sodium azide or bromoheptane (a GSH-depleting agent). Cell viability was determined by trypan blue uptake. Similar experiments were undertaken for CGA and DHCA. Three separate experiments were carried out. Values shown are means ± S.E.
↵5-150 Data were significantly different from the control (P < 0.05). g.o., glucose oxidase.
↵5-a The rate of hydrogen peroxide generated by the glucose/glucose oxidase system used was 40 nmol/ml/min, which had no toxicity effect on hepatocytes (determined using a Clark type oxygen electrode).
↵5-b None of the inhibitors (benzylimidazole, dicumarol, sodium azide), hydrogen peroxide-generating system (glucose/glucose oxidase system), hydroperoxides, and tyrosinase were toxic at the dose given alone (data not shown).