Trout DEHP metabolism

The metabolism of C-DEHP by rainbow trout liver subcell-ular fractions and serum was studied by Melancon and Lech (14). The data in Table VI show that without added NADPH, the major metabolite produced was mono-2-ethylhexyl phthalate. When NADPH was added to liver homogenates or the mitochondrial or microsomal fractions, two unidentified metabolites more polar than the monoester were produced. Additional studies showed that the metabolism of DEHP by the mitochondrial and the microsomal fractions were very similar (Figure 1). Both fractions show an increased production of metabolites of DEHP resulting from addition of NADPH and the shift from production of monoester to that of more polar metabolites. The reduced accumulation of monoester which accompanied this NADPH mediated production of more polar metabolites may help in interpreting the pathway of DEHP metabolism in trout liver. This decreased accumulation of monoester could be explained either by metabolism of the monoester to more polar metabolites or the shift of DEHP from the hydrolytic route to a different, oxidative pathway. The latter explanation is unlikely because in these experiments less than 20% of the DEHP was metabolized. 

Because the metabolism of DEHP was catalyzed by so many fractions of the trout liver homogenate, these fractions were characterized by measurement of marker enzymes to determine which organelles actually were responsible for the observed DEHP metabolism. Succinic dehydrogenase activity was used as a marker for mitochondria, whereas glucose-6-phosphatase was used as a marker for microsomes. The distribution of DEHP oxidase activity (production of polar metabolites 1 and 2 with added NADPH) and of DEHP esterase activity (production of monoester without added NADPH) were also determined. It was found (Figure 2) that the distribution of DEHP oxidase activity parallels the distribution of microsomal activity and the distribution of DEHP esterase activity parallels the distribution of microsomal activity, but is also present in the cytosol fraction. 

Figure 2. Distribution of marker enzymes and DEHP-metabolizing enzymes in trout liver homogenate fractions. DEHP esterase and DEHP oxidase were each measured by 1-hr incubations of 0.010 ftmol of UC-DEHP in a total volume of 2 mL. Fraction (A), 2,000 g pellet (B), 10,000 g pellet (C), 100,000 g pellet and (D), 100,000 g supernatant. Relative Specific Activity = percent of total activity/ percent of total protein (14).

Although the metabolism of several phthalate esters has been studied in vitro, essentially all of the in vivo studies have involved DEHP. A summary of these experiments which involved exposure offish to aqueous - C-DEHP is presented in Table IV (11,12). Tissue C was isolated and separated into parent and the various metabolites by preparative thin layer chromatography on silica gel. Metabolites were hydrolyzed where appropriate and identified by gas chromatography-mass spectroscopy. In whole catfish, whole fathead minnow and trout muscle, the major metabolite was the monoester while in trout bile the major metabolite was the monoester glucuronide. The fact that in all cases the major metabolite was monoester or monoester glucuronide despite the differences in species, exposure level and duration, etc. represented by these data, suggests that hydrolysis of DEHP to monoester is important in the biotransformation of DEHP by fish. 

In an effort to characterize further the metabolism of DEHP by trout, the effect of the mixed function oxidase inhibitor, piperonyl butoxide, upon the metabolism of DEHP by these trout liver fractions and serum was examined. Because of the use of piperonyl butoxide as an insecticide synergist, it is possible that fish might be exposed to this chemical in the environment. The data in Table VII show that piperonyl butoxide inhibited overall metabolism of DEHP by liver homogenates and microsomes whether NADPH was added or not. The hydrolysis of DEHP by serum was also blocked by piperonyl butoxide and although not shown, this was also the case with liver cytosol. These latter results were surprising because piperonyl butoxide has been known as a mixed function oxidase inhibitor only, and would not be expected... 

Figure 1. Influence of time on metabolism of I4C-DEHP by trout liver mitochondrial and microsomal fractions. Incubation contained 0.010 /imol of 14C-DEPH in a total volume of 2 mL. Mitochondria equivalent to 0.254 g of liver or microsomes equivalent to 0.361 g of liver were used in each incubation. Open bars represent monoester, striped bars Polar Metabolite I and solid bars Polar Metabolite 2. Each column represents an individual incubation (14).

Because the metabolism of DEHP was relatively slow, the more readily hydrolyzed 2,4-dichlorophenoxyacetic acid-n-butyl ester was sometimes used for comparison. The hydrolysis of this compound, both by liver preparations and by serum also was inhibited by piperonyl butoxide. Liver homogenates from trout, which had been exposed to piperonyl butoxide in vivo, showed decreased capacity to metabolize DEHP (Table VIII). 

In Vivo exposure of trout to piperonyl butoxide also affected the disposition and metabolism of l C-DEHP. The results in Table IX show that piperonyl butoxide reduced biliary but increased ll+C in muscle and blood. Because the bile contains mostly DEHP metabolites, this represents decreased metabolism. 

In additional in vitro studies with a number of methylene-dioxyphenyl compounds, only tropital, in addition to piperonyl butoxide, had similar inhibitory effects on the metabolism of DEHP by trout liver homogenates and serum (16). Of the methyl-enedioxyphenyl compounds studied, only tropital had a long side chain like that of piperonyl butoxide. This suggests that similarities in the side chains of these two compounds and the side chains of DEHP and 2,4-dichlorophenoxyacetic acid-n-butyl ester may be responsible for this inhibition. 

It was also found that paraoxon, an esterase inhibitor, substantially reduced formation of polar metabolite 1 from DEHP by trout liver microsomes with added NADPH. This suggests that polar metabolite 1 is formed via further metabolism of the monoester, the production of which was reduced by paraoxon. 

Fish liver microsomes are capable of both hydrolytic and oxidative metabolism of phthalate esters. In addition, trout liver cytosol and blood serum exhibited esterase activity against DEHP. 

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See also in sourсe #XX -- [ ]

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