2,3,7,8-tetrachlorodibenzo-p-dioxin alters melatonin metabolism in fish hepatocytes

Pineal hormone melatonin is an important regulator of endocrine and circadi an rhythms in vertebrates. Since liver is assumed to be the major organ in the metabolism of this indole hormone, we investigated the effect of the kn own Ah-receptor agonist. 2,3,7,8-tetra-chlorodibenzo-p-dioxin (TCDD) on mel atonin metabolism in fish hepatocytes as well as the in vitro effect of mel atonin on trout hepatic microsomal cytochrome P4501A (CYP1A) catalyst. Prim ary cell cultures of rainbow trout hepatocytes were exposed to [H-3]melaton in (1 nM to I mu M) alone and in combination with TCDD (50 pM) at 15 degree s C for 24 or 48 h. Analysis of melatonin and its metabolites in the cultur e medium and hepatocytes by HPLC revealed that about 96% of the added [H-3] melatonin was metabolised after 24 h in both control and TCDD treated cultu res. H-3-radioactivity was found mainly in the culture medium and less than 5% of the total H-3-radioactivity retained inside hepatocytes. Of the HPLC separated metabolites, one coeluted with 6-hydroxymelatonin and one unknow n metabolite eluted after 6-hydroxymelatonin. In addition, two other metabo lites were more water-soluble than 6-hydroxymelatonin and were considered t o be conjugated products. Treatment of the hepatocytes with TCDD increased the amount of the major oxidated product, 6-hydroxymelatonin, about 2.5-fol d after 24 h and 1.2-fold after 48 h exposure, respectively when compared w ith the control cultures. Whereas the amount of the unknown metabolite duri ng after 6-hydroxymelatonin decreased about 1.3-fold after 24 h and 1.2-fol d after. 48 h exposure. respectively. Melatonin alone did not affect P4501A associated EROD-activity or CYP1AmRNA levels in the primary hepatocyte cul tures. TCDD-treatment increased EROD-activity 3 to 5-fold and respective CY P1AmRNA content 6 to 14-fold, when compared with the control or melatonin-t reated cultures. Furthermore, melatonin competitively inhibited EROD-activi ty in liver microsomes with a Ki value of 62.06 +/- 3.78 mu M. The results show that TCDD alters metabolic degradation of melatonin in hepatocytes and suggest that P4501A may be an important P450 isoenzyme involved in oxidati ve metabolism of melatonin in fish liver. (C) 3000 Elsevier Science Ireland Ltd. All rights reserved.