Skate hepatic microsomes

Several properties of hepatic microsomal AHH activity were compared in control and DBA-pretreated male little skates as shown in Table I. Following treatment there was an approximately 35-fold increase in specific enzyme activity, as quantitated by fluorescence of the phenolic metabolites formed (3, 21). The pH optimum, which was fairly broad, and the concentration of benzo(a)-pyrene (0.06 mM) that had to be added to the incubation mixture to achieve maximum enzyme activity were the same for both control and induced skate hepatic microsomes. The shorter periods observed for linearity of product formation with microsomes from the induced skates is thought to be related to the much higher AHH activity present, and may be due to substrate depletion or the formation of products which are inhibitory (i.e., compete with the MFO system as they are substrates themselves). A similar explanation may be relevant for the loss of linear product formation at lower microsomal protein concentrations in the induced animals. 

Hepatic microsomal and solubilized mixed-function oxidase systems from the little skate, Baja erinacea, a marine elasmobranch. In Ullrich, V., Hildebrandt, A., Roots, I., Eastabrook, R.W. (Eds.) Microsomes and Drug Oxidations (1976). Pergamon Press, Oxford, pp 16O-I69. 

In general, our studies with cytochrome P-450-dependent metabolism have emphasized the similarity of the hepatic MFO system in marine fish to that found in mammals. Thus, in the little skate (Raja erinaoea), a marine elasmobranch, enzyme activity is localized in the microsomal fraction, requires NADPH and molecular oxygen for maximum activity, and can be inhibited with CO (1, 2). Moreover, when hepatic microsomes from the little skate were solubilized and separated into cytochrome P-450, NADPH-cytochrome P-450 reductase, and lipid fractions, all three fractions were required for maximal MFO activity in the reconstituted system (3). We have also found, as have others, that the administration of polycyclic hydrocarbons (3-methylcholanthrene, 1,2,3,4-dibenzanthracene [DBA]), 2,3,7,8-tetrachlorodibenzo-p-dioxin... 

In this report we compare several properties of hepatic microsomal AHH activity in control and DBA-treated little skates (including metabolic profiles obtained from c-benzo(a)pyrene as elucidated by high pressure liquid chromatography [HPLC]), we describe the partial purification of two different forms of cytochrome P-450 (cytochrome P-448 and cytochrome P-451) from hepatic microsomes of DBA-pretreated little skates and we report polycyclic hydrocarbon-like induction in large numbers of winter flounder assayed in Maine during June, July, and August, which was quite different than data obtained with sheepshead examined in Florida during the same period. 

Solubilization and Partial Purification of Cytochrome P-450 from Hepatic Microsomes of Male, DBA-Pretreated Little Skates. Washed hepatic microsomes (3) from the livers of 10 skates were suspended in 0.25 M sucrose and frozen under nitrogen (-5 to -10°) at the Maine laboratory. They were then packed in dry ice and transported to NIEHS, Research Triangle Park, NC, within 14 days of preparation and were stored at -62°C until use. Microsomes... 

The apparent kinetic constants were obtained from Lineweaver-Burk plots of AHH activities recorded in the presence of increasing concentrations of benzo(a)pyrene (0.001-1.0 mM). The plots were linear for both untreated and DBA-induced animals. The apparent V was 20- to 30-fold higher in hepatic microsomes from the induced skates whereas the apparent K values were of the same magnitude in control and treated fish. 

An obvious difference was also noted between control and induced skate hepaticdnicrosomal AHH activity in the presence of a-naphthoflavone (10 M). This compound, when added in vitro at this or higher concentrations, caused significant stimulation of AHH activity in control animals (about 3-fold) but inhibition (80%) was found in DBA-pretreated skates. Similar results were earlier reported for control and 3-methylcholanthrene-treated rats (23), where it appears that the response is due to differential effects of a-naphthoflavone on hepatic microsomal cytochrome P-450 (stimulated) and cytochrome P-448 (inhibited) (24). Our data suggests that there may be a novel form of cytochrome P-450 synthesized in skate liver in response to polycyclic hydrocarbon administration, even though there was no hypsochromic shift in the carbon monoxide difference spectrum of dithionite reduced hepatic microsomes from DBA-treated skates (relative to hepatic microsomes from control fish). 

The elution profile of cytochrome P-448 (absorption at 418 nm) and epoxide hydratase activity from a sodium cholate-solubi-lized hepatic microsomal preparation (from DBA-treated male skates) applied to a DEAE-cellulose column and eluted with Buffer II is shown in Fig. 3. The void volume of the column contained significant amounts of epoxide hydratase activity. Fractions 40-70 (Fig. 3) were combined, and concentrated. The carbon monoxide difference spectrum, which had an absorption maximum at 448 nm in the induced state, is shown in Fig. 4. This form of the cytochrome (i.e.,... 

Figure 1. Production of total BP metabolites by hepatic microsomes from control and DBA-pretreated male little skates. Each point is the result of a single incubation and HPLC determination (O----O), control skates ( — ), DBA-pre-...

Figure 2. Profile of radioactive metabolites obtained upon incubation of UC-BP with hepatic microsomes from control or DBA-pretreated male little skates.

Figure 3. Elution profile of partially purified Cytochrome P-448 and epoxide hydratase activity of solubilized hepatic microsomes from DBA-treated male skates from a DEAE-cellulose column with Buffer II. Epoxide hydratase activity was determined with BP-4,5-oxide as the substrate (see Materials and Methods).

Figure 4. Carbon monoxide difference spectrum of partially purified hepatic microsomal Cytochrome P-448 from DBA-treated little skates. The cuvettes contained dithionite-reduced cytochrome (0.10 mg protein/mL) in lOmM phosphate buffer, pH 7.7, containing 20% glycerol, O.lmM EDTA and O.JtnM dithiothreitol.

Very similar results to those described in Fig. 3-6 were obtained when sodium cholate solubilized hepatic microsomes from DBA-treated female little skates were subjected to chromatography on DEAE-cellulose as described above (data not shown). Also not shown are the results obtained with hepatic microsomes from untreated male and female little skates. With untreated animals, 80-90% of the cytochrome P-450 eluted from the DEAE-cellulose column only at higher ionic strength (i.e., with the KC1 gradient). However, in all preparations studied, an appreciable amount of cytochrome P-450 (10-20%), having its absorption maximum in the carbon monoxide-ligated and reduced state at 450 nm, was eluted from the column with buffer II, as was observed with cytochrome P-448 of hepatic microsomes from DBA-treated skates. The further purification of the various forms of cytochrome P-450 from control and DBA-pretreated little skate livers is currently in progress in our laboratory. 

Collectively, these experiments have demonstrated that substantial amounts of cytochrome P-448 are indeed present in liver of DBA-pretreated male and female skates but not in untreated skates. Sufficient cytochrome P-448 is present to account for the increased AHH activities observed in hepatic microsomes of DBA-treated skates. 

One of our more interesting observations is illustrated in Table III. The administration of DBA to winter flounder increased hepatic microsomal AHH and 7-ethoxyresorufin deethylase activities as expected, and AHH activity was strongly inhibited in the DBA-treated flounder by 10" M a-naphthoflavone as we have previously reported for both little skate (4) and sheepshead (9). However, the presence of high AHH and 7-ethoxyresorufin deethylase activities in one control flounder, and the inhibition of AHH activity by a-naphthoflavone in this animal suggested that the hepatic microsomal MFO system of this fish was already induced. 

Bend, J. R., Hall, P., and Foureman, G. L. Comparison of benzo(a)pyrene hydroxylase (aryl hydrocarbon hydroxylase, AHH) activities in hepatic microsomes from untreated and 1,2,3,4-dibenzanthracene (DBA)-induced little skate (Raja erinacea). Bull. Mt. Desert Island Biol. Lab. (1976) 16. 3-5. 

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). 

Pohl, R.J., J.R. Fouts and J.R. Bend. Response of hepatic microsomal mixed-function oxidases in the little skate, Raja erinacea, and the winter flounder, Pseudopleuronectes americanus to pretreatment with TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) or DBA (1,2,3,4-dibenzanthracene). Bull. Mt Desert Isl. Biol. Lab. 15 64-66, 1975. 

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