Hemoprotein 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). 

This initial study demonstrating induction of monooxygenation and hemoprotein P-450 in the rainbow trout by polycyclic aromatic hydrocarbons, but not by phenobarbital, was extended further. 

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.

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). 

In summary, it would appear that rainbow trout are responsive to cytochrome P -450-type inducers (e.g. planar polychlorinated biphenyls and polycyclic aromatic hydrocarbons), but are not responsive to cytochrome P-450-type inducers (e.g. phenobar-bital and non-planar polychlorinated biphenyls). Although induced rainbow trout hemoprotein(s) P-450 show high activity with cytochrome P -450 substrates, the induced hemoprotein at 57,000 daltons appears, in electrophoretic and spectral properties, to differ from rodent cytochrome P -450. 

Elcombe, C.R. and Lech, J.J. Induction and characterization of hemoprotein(s) P-450 and monooxygenation in rainbow trout (Salmo gairdneri). Toxicol. Appl. Pharmacol. (1979) In Press. 

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