Trout microsomes

Microsomes are widely used to study the metabolism of xenobiotics. Enzymes can be chararacterized on the basis of their requirement for cofactors (e.g., NADPH, UDPGA), and their response to inhibitors. Kinetic studies can be carried out, and kinetic constants determined. They are very useful in studies of comparative metabolism, where many species not available for in vivo experiment can be compared with widely investigated laboratory species such as rats, mice, feral pigeon, Japanese quail, and rainbow trout. 

Pesonen, M., Goksoyr, A., and Andersson, T. (1992). Expression of P450 lAl in a primary culture of rainbow trout microsomes exposed to B-naphthoflavone or 2,3,7,8-TCDD. Archives of Biochemistry and Biophysics 292, 228-233. 

Dady JM, SP Bradbury, AD Hoffman, MM Voit, DL Olson (1991) Hepatic microsomal Al-hydroxylation of aniline and 4-chloroaniline by rainbow trout (Oncorhyncus mykiss). Xenobiotica 21 1605-1620. 

Vodicnik, M.J., C.R. Elcombe, and J.J. Lech. 1981. The effect of various types of inducing agents on hepatic microsomal monooxygenase activity in rainbow trout. Toxicol. Appl. Pharmacol. 59 364-374. 

Melancon, M.J., K.A. Tumquist, and J.J. Lech. 1989. Relation of hepatic microsomal monooxygenase activity to tissue PCBs in rainbow trout (Salmo gairdneri) injected with [14C] PCBs. Environ. Toxicol. Chem. 8 777-782. 

Sediments and biota collected from the Hersey River, Michigan, in 1978, were heavily contaminated with phenanthrene, benz[a]anthracene, and benzo[a]pyrene when compared to a control site. Elevated PAH concentrations were recorded in sediments, whole insect larvae, crayfish muscle, and flesh of lampreys (family Petromyzontidae), brown trout (Salmo trutta), and white suckers (Catostomus commersoni), in that general order (Black et al. 1981). The polluted collection locale was the former site of a creosote wood preservation facility between 1902 and 1949, and, at the time of the study, received Reed City wastewater treatment plant effluent, described as an oily material with a naphthalene-like odor (Black et al. 1981). In San Francisco Bay, elevated PAH concentrations in fish livers reflected elevated sediment PAH concentrations (Stehr et al. 1997). In Chesapeake Bay, spot (Leiostomus xanthurus) collected from a PAH-contaminated tributary (up to 96 mg PAHs/kg DW sediment) had elevated cytochrome P-450 and EROD activity in liver and intestine microsomes (Van Veld et al. 1990). Intestinal P-450 activity was 80 to 100 times higher in fish from highly contaminated sites than in conspecifics from reference sites intestinal EROD activity had a similar trend. Liver P-450 and EROD activity was about 8 times higher in spot from the contaminated sites when compared to the reference sites. Liver P-450 activity correlated positively with sediment PAH, but intestinal P-450 activity seemed to reflect dietary exposure (Van Veld et al. 1990). The poor correlation between hepatic concentrations of PAHs and P-4501A is attributed to the rapid metabolism of these compounds (van der Weiden et al. 1994). 

To acquire information on the intrinsic metabolic activity of aquatic organisms, liver of carp (Cyprinus carpio Linnaeus), rainbow trout (Salmo gairdneri) and freshwater snail (Cipango-paludina japonica Martens) was dissected out, homogenized in 0.1M phosphate buffer, pH 7.5, and centrifuged at 105,000 g for 60 min to obtain the microsome-equivalent (described as the microsomal fraction hereafter) fraction. The protein content of microsomal and submicrosomal (supernatant fractions by Lowry s method, microsomal P-450 content ( ), activity of aniline hydroxylase (4) and aminopyrine N-demethylase (5) were determined. 

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. 

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).

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. 

Table I). The levels of both, cytochrome P-L50 (Table i) and its NADPH (reduced nicotinamide adenine dinucleotide phosphate) requiring reducing component (Figure l)(which can be measured as NADPH dependent cytochrome c reductase) are substantial in fish liver microsomes, although lower than in mammals. NADPH cytochrome c reductase level in trout Salmo trutta lacustris) is 20 nmol cytochrome c reduced/mg microsomal protein/min the corresponding activity in male Sprague Dawley rat liver microsomes is 96 nmol cytochrome c reduced/mg microsomal protein/min (lU). 

Cytochrome P-450 spectra (reduced + CO) show the 2-nm shift to the blue as a result of 3-MC induction. No such shift is observed in the trout, control rat, and PB-induced rat liver microsomes. Cytochrome P-450 EtNC spectra were recorded at pH 7.4, 2 min after the samples were reduced with dithionite. The absorption peaks are at 430 and 455 nm. For pH curves the A Absorbance represents A A (430—490 nm) and A A (455— 490 nm). The number of (+) signifies only the relative activity or inhibition with respect to BP hydroxylation and covalent binding of BP to DNA ( ++-(-) signifies 2-4 times and (+-H—h) signifies 10-36 times the control rat microsomal activity (n.d.), not determined. 

It has been noted that cell cultures derived from trout (66, 67) and trout liver microsomes (U2, U7, 68) may have relatively high AHH activity (BP hydroxylase) which sometimes exceeds the activity observed in control rat liver microsomes. The metabolite pattern obtained using trout liver microsomes resembled that produced by MC treated rat liver microsomes (P-Uh8)(6 and Ahokas, Saarni, Nebert and Pelkonen, manuscript in preparation Table IV). Associated with this pattern of BP metabolites was covalent binding to DNA which was three times as high as obtained by using rat liver microsomes. Another species of fish (roach), on the other hand was found to be almost inactive in catalyzing in vitro bind-... 

Figure 3. Preferential routes of metabolism of 2-AAF, biphenyl, and BP (from top to bottom) by different forms of rat liver microsomal Cytochrome P-450. Trout has been reported to metabolize 2-AAF (65) and biphenyl (37) like rat liver Cytochrome P-450 and BP like rat liver Cytochrome P-448 (69,70).

P-1+50 and drug-induced spectral interactions in the hepatic microsomes of trout, Salmo trutta laoustvis. Acta Pharmacol. et Toxicol. (1976) 38, 1+1+0—1+1+9-... 

Chan, T.M., Gillett, J.W. and Terriere, L.C. Interaction between microsomal electron transport systems of trout and male rat in cyclodiene epoxidation. Comp. Biochem. Physiol. (1967), 20, 731-7 +2. 

Ahokas, J.T. Metabolism of 2,5-diphenyloxazole (PP0) by trout liver microsomal mixed function monooxygenase. Res. Commun. Chem. Pathol. Pharmacol. (1976) 13, 1+39-1+1+7. 

The in vitro metabolism and covalent binding of benzo(a) pyrene to DNA catalysed by trout liver microsomes. 

Benzo(a)pyrene is converted by the microsomal MFO system of mammals (18) and trout (25) to a number of oxidized products. 

Induction of Hepatic Microsomal Enzymes in Rainbow Trout... 

Arylhydrocarbon (benzo[a]pyrene) hydroxylase, benzphetamine-N-demethylation, ethylmorphine-N-demethylation, ethoxycoumarin-0-deethylation and ethoxyresorufin-0-deethylation were performed by published procedures (31,32,33,34), but optimized for use with trout microsomes as described previously (30, 35). Hemoprotein P-450 was determined by the procedure of Estabrook et al. (36) to avoid spectral interference by hemoglobin. Microsomal protein content was estimated either by the method of Ross and Shatz (37) or Lowry et al. (38), using bovine serum albumin standards. 

Initial studies designed to obtain a valid subcellular fractionation scheme for rainbow trout liver illustrated the aryl-hydrocarbon (benzo[a]pyrene] hydroxylase activity separated with glucose-6-phosphatase (35). This observation indicated that the trout hemoprotein P-450-mediated monooxygenation system was located within the endoplasmic reticulum (microsomal fraction). 

Table II demonstrates the effect of two polychlorinated biphenyl mixtures (Aroclors 1254 and 1242), a polybrominated biphenyl mixture (Firemaster BP6), phenobarbital and -naphtho-flavone on various hemoprotein P-450-mediated monooxygenase activities of rainbow trout hepatic microsomes.

Figure 1. Effect of potential inducing agents on certain hepatic microsomal enzymes of the rainbow trout. Animals were infected intraperitoneally with either phenobarbital (65 mg/kg) 3-methylcholanthrene (20 mg/kg) 2,3-benzanthracene (10 mg/kg) or /3-naphthoflavone (100 mg/kg). The animals were sacrified and hepatic microsome prepared 48 hr after infection. Each bar is the mean SE (n = 3-5) ( ), induced activity (T) significantly different from control (C) activity...

The alteration of hemoprotein(s) P-450 subpopulations in the rat may be observed spectrally, because after treatment of rats with polycyclic aromatic hydrocarbons, the Soret maximum of the carbonmonoxyferrocytochrome complex undergoes a hypsochromic shift from 450 to 448nm (50). This blue shift was not seen with rainbow trout hepatic microsomes (29,30). However, this does not preclude the induction of novel hemoproteins P-450 since (a) the induced hemoprotein(s) maty not differ spectrally from the constitutive enzymes and (b) the induced-hemoprotein may account for only a small proportion of total hemoprotein P-450, and hence its contribution to the position of the Soret maximum of carbon monoxide-treated reduced microsomes may be negligible. The latter suggestion is supported by the work of Bend et al. with the little skate. These workers have shown that hepatic microsomes from 1, 2,3,4-dibenzanthracene treated skates did not exhibit a hypsochromic shift when compared to control microsomes, however, partially purified hemoprotein exhibited an absorbance maxima at 448 nm (51). 

Figure 3. Spectra of ethylisocyanide-ferrocytochrome P-450 complexes, rainbow trout. Conditions were as in Figure 2, except rainbow trout hepatic microsomes were used. (A) Microsomes from rainbow trout treated with (j-naphthoflavone (100 mg/kg sacrificed 4 days later). Protein concentration, 1.4 mg/mL total P-450 concentration, 0.48 fi.M. (B) Microsomes from control fish. Protein concentration, 0.93 mg/mL, total P-450 concentration, 0.22 /iM.

Figure 4. SDS-polyacrylamide gel electrophoresis of microsomes from variously pretreated rainbow trout (A), control microsomes, 90 fig protein/gel (B), /3-naphthoflavone-inauced microsomes, 90 fig protein/gel (C), Aroclor 1242-induced microsomes, 90 fig protein/gel.

Induction of Hepatic Microsomal Monooxvgenation in the Rainbow Trout by Selected Polychlorinated Biphenyls... 

Figure 5. Densitiometric scans of electrophoretograms of hepatic microsomes from rainbow trout pretreated with polychlorinated biphenyl congeners (A), control microsomes, 90 fig protein/gel (B), 2,2, 4,4 -tetrachlorobiphenyl-induced microsomes, 90 fig protein/gel (C), 3,3, 4,4 -tetrachlorobiphenyl-induced microsomes, 90 fig protein/gel (D), Aroclor 1242-induced microsomes, 90 fig protein/ gel. The slab gels were stained with Coomassie Blue-250 and individual sample tracts were cut out and scanned at 550 nm. The vertical broken line is at 57,000...

Statham, C.N., Elcombe, C.R., Szyjka, S.P. and Lech, J.J. Effect of polycyclic aromatic hydrocarbons on hepatic microsomal enzymes and disposition of methylnaphthalene in rainbow trout in vivo. Xenobiotica. (1978) j3 65-71. 

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