Little skate

Silverside, inland, Menidia beryllina, 594, 604 Skate, little. Raja erinacea, 269, 418 Smelt, rainbow, Osmerus mordax, 512 Snakehead, Channa sp., 104, 147 Sole... 

Recently Bend et al. have succeeded to solubilize and partially purify little skate liver microsomal cytochrome P-l+50 and have thus been able to record the absolute spectra of fish cytochrome P-l+50 (25.). The spectra are very similar to those of rat liver microsomal cytochrome P-l+50 and purified rat liver cytochrome P-1+50 and P-1+1+8 (Table II). 

Table II. Comparison of the spectral properties of partially purified little skate liver cytochrome P-l+50 with partially purified rat liver cytochrome P-l+50 and highly purified rat liver cytochro-me P-l+50 and P-1+1+8. ...

Partially purified Little Skate liver cytochrome P-l+50. The data is from (25.) and is as accurate as can be read from published... 

Bend, J.R., Pohl, R.J., Davidson, N.P. and Fouts, J.R. Response of hepatic and renal microsomal mixed-function oxidases in the little skate, Ra.ja erinacea, to pretreatment with 3-methyl-cholanthrene or TCDD (2,3,7,8-tetrachlorodibenzo- -dioxin). Bull. Mt, Desert Is. Biol. 

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. 

Fish Collection, Maintenance, and Treatment. Adult fish were collected near Mount Desert Island, Maine, or Marineland, Florida, and were acclimated in aquaria equipped with continuously flowing seawater or in live cars immersed in salt water for at least 24 hr before use. For induction studies little skates were injected IP with 10 mg/kg 1,2,3,4-dibenzanthracene in corn oil on days 1, 2, and 3 and were sacrificed on day 10. Control fish were injected with corn oil only. 

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... 

Thawed microsomal preparations (500-700 mg protein) from little skates were digested with sodium cholate (1 mg/mg protein) in 10 mM potassium phosphate buffer (pH 7.7) to make a final concentration of 10 mg protein/ml, in the presence of 0.1 mM EDTA,... 

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. 

DBA-PRETREATED MALE LITTLE SKATES (Raja erinacea)... 

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 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. 

In spite of these limitations there seems little doubt that induction of the hepatic MFO system in certain fish can indicate the presence of selected toxic chemicals (to fish and humans) in both freshwater and marine environments. For this reason we have been studying various aspects of this question for the last few years in freshly captured fish (winter flounder and little skate in Maine and the sheepshead in Florida) and in fish induced by polycyclic hydrocarbon administration. 

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., Pohl, R. J., and Fouts, J. R. Some properties of the microsomal drug-metabolizing enzyme system in the little skate, Rada evinaoea. Bull. Mt. Desert Island Biol. Lab. (1972) 12 9-12. 

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). 

Bend, J.R., R.J. Pohl and J.R. Fouts. Further studies of the microsomal mixed-function oxidase system of the little skate, Raja erinacea, including its response to some xenobiotics. Bull. Mt Desert Isl. Biol. Lab. 13 9-13, 1973. 

Merson, R.R., D. Gilbert, J.J. Stegeman and M.E. Hahn. Xenobiotic metabolizing enzymes in elasmobranchs cloning of the cytochrome P450-1A (CYP1A) cDNA in little skate Raja erinacea. Mar. Environ. Res. 58 538-539, 2004. 

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