Induction of monooxygenation in rainbow trout by polybrominated biphenyls: a comparative study.

Two commercial polychlorinated biphenyl mixtures (Aroclor 1254 and Aroclor 1242) and one polybrominated biphenyl mixture (FireMaster BP-6) were examined for their abilities to induce hepatic microsomal monooxygenation in rainbow trout (Salmo gairdneri). Pretreatment of rainbow trout with Aroclors 1254 and 1242 (150 mg/kg IP) resulted in an approximate 10-fold induction of arylhydrocarbon (benzo[a]pyrene) hydroxylation, ethoxycoumarin-O-deethylation and ethoxyresorufin-O-deethylation within 7 days after injection. These enzyme activities remained elevated above control values for at least 2-3 weeks. Administration of FireMaster BP-6 (150 mg/kg IP) also resulted in an induction of several monooxygenase activities. Arylhydrocarbon (benzo[a]pyrene) hydroxylation, ethoxycoumarin-O-deethylation and ethoxyresorufin-O-deethylation were increased by 6-, 3,- and 25-fold, respectively. Only the latter two activities remained elevated two weeks post-injection. Ethylmorphine-N-demethylation was unaffected by the polyhalogenated biphenyls. Significant increases in P-450 hemoprotein were not observed after pretreatment with any of the polyhalogenated biphenyls studied.


Introduction
The polyhalogenated biphenyls are well recognized as toxic environmental contaminants (1)(2)(3)(4)(5)(6), and the polychlorinated biphenyls (PCBs) have been extensively studied as inducers of hepatic microsomal monooxygenation in several species (7)(8)(9)(10)(11)(12); however only recently have reports appeared indicating the inducing potential of the polybrominated biphenyls (PBBs) (13)(14)(15)(16). These studies have suggested that the PCBs and PBBs are agents which exhibit the induction properties of both the barbiturate and the polycyclic aromatic hydrocarbon classes of inducers. Classically t Although we realize this terminology is an over simplification, we shall use the name cytochrome P-450 for the monooxygenase hemoprotein(s) induced by the phenobarbital type of agents and cytochrome P,-450 for those induced by the polycyclic aromatic hydrocarbon type of agents. To indicate a nonspecified cytochrome which when reduced and complexed with CO exhibits an absorption maximum around 450 nm, we shall use the term hemoprotein P-450 (17). phenobarbital is a representative member of a class of agents which induce cytochrome P-450, while 3-methylcholanthrene and other polycyclic aromatic hydrocarbons induce cytochrome P1-450 (P-448).t Aquatic animals hold an important position in the food chain. Fish are capable of accumulating lipid soluble compounds from their environment leading to large bioconcentration factors (18).
Both PBBs and PCBs have been shown to be accumulated in fish tissues (19,20), and 2,5,2',5'tetrachlorobiphenyl was shown to have a half-life of elimination from rainbow trout of 2.66 years (21). Because of the resistance of PBBs and PCBs to metabolic degradation and hence their long halflives it was of interest to study their induction properties in fish.
Despite initial suggestions to the contrary, it is now recognized that fish possess drug metabolism mechanisms which are similar to those of mammals (22)(23)(24)(25)(26). Furthermore, induction of monooxygenation reactions by environmental pollutants, such as the polycyclic aromatic hydrocarbons (27)(28)(29) and PCBs (30,31) have been reported. It has been proposed that field measurements of hepatic arylhy-drocarbon hydroxylase in fish species may be used as an indicator of aquatic pollution due to petroleum (32).
Although several similarities between mammalian and fish microsomal hemoprotein P-450-mediated monooxygenation exist, a fundamental difference in induction mechanisms has been observed. Although polycyclic hydrocarbons are capable of inducing monooxygenation; inducers of the barbiturate class (e.g. phenobarbital, phenylbutazone, and DDT) are unable to stimulate monooxygenase activity in several species of fish (23,33,34).
As previously described, PCBs and PBBs produce a "mixed" type of induction in mammals. Hence, since the mechanism for phenobarbital type induction appears to be absent in fish, this species may be a suitable animal model for study of the induction of P1-450-like enzymes by polyhalogenated biphenyls.

Materials and Methods
Rainbow trout (Salmo gairdneri), weighing 70-100 g, were obtained from Kettle Moraine Springs Trout Hatchery (Adell, Wisconsin), and were held in flowing, charcoal filtered water 12°C for a minimum of two weeks prior to use. A 12-hr (6 AM-6 PM) light cycle was operated.
NADP, NADPH NADH, glucose-6-phosphate, glucose-6-phosphate dehydrogenase and 7hydroxycoumarin (umbelliferone) were obtained from Sigma Chemical Company (St. Louis, Missouri). 7-Ethoxycoumarin was synthesized by a published method (35 Fish were sacrificed by a blow to the head, and the gall bladders were carefully removed. The livers were excised and rinsed in ice-cold 0. 154M KCI to remove adhering hemoglobin. The livers were minced and washed three times with 0.154M KCI. After washing, the minced livers were homogenized in 4 volumes of 0.25M sucrose by using a motor-driven Potter-Elvehjen-type glass-Teflon homogenizer. The liver homogenates were centrifuged at 8500 g (rav = 8.3 cm) for 20 min by using a Sorval type 24 rotor in a Sorval RC-5 Superspeed centrifuge. The resultant supernatant was decanted and centrifuged at 165,000g (rav = 5.7 cm) for 60 min by using a Beckman type 65 rotor in a Beckman L6-65 ultracentrifuge. The microsomal pellet obtained was resuspended in 0.154M KCI and recentrifuged at 165,000g for 60 min. The washed pellet was suspended in 0.25M sucrose to a concentration equivalent to 1 g wet weight of liver/ml of suspension.
All operations were performed at 0-40C, and the microsomes were utilized on the day of preparation.
Microsomal protein was measured by the procedure of Ross and Schatz (40) by using crystalline bovine serum albumin standards.

Spectral Studies
Cytochrome P-450 was estimated by the difference in absorbance between the CO complex of Na2S204 reduced microsomes and oxidized microsomes by using an extinction coefficient of 100 mM-1-cm-' between 450 and 510 nm (41). Ethylisocyanide-reduced difference spectra were obtained by the method of Imai and Sato (42). All spectra were obtained at room temperature with a Cary-Varian 219 spectrophotometer.

Results
Pretreatment of rainbow trout with the polyhalogenated biphenyls (FireMaster BP-6, Aroclor 1242, or Aroclor 1254) had no effect on the liver/body ratios or yield of microsomal protein per unit wet weight of liver (Table 1)     Ethylmorphine-N-demethylation was unaffected by pretreatment of fish with Aroclors 1254 or 1242. This is illustrated for Aroclor 1254 in Figure 4.
After injection of rainbow trout with FireMaster BP-6 no increases in monooxygenase activities were seen until 3 days after treatment (Fig. 5). However, at this time AHH, ethoxyresorufinand ethoxycoumarin-O-deethylations were elevated. AHH declined rapidly to reach control levels about 7 days after treatment of the fish, while the latter two activities remained elevated for at least 2 weeks. In common with the PBCs, PBBs also failed to increase ethylmorphine-N-demethylation above control values.
The stimulatory effect of PCBs and PBBs upon monooxygenations appears to be a true induction, since inclusion of these compounds in the in vitro assays had no effect upon the observed enzymatic activities.
Hemoprotein P-450 concentrations in rainbow trout hepatic microsomes were determined 4 days after pretreatment with various inducing agents. Small increases (10-20%) in hemoprotein P-450 content were observed after treatment of fish with PCBs or PBBs, but these increases were not statistically significant (Table 2). However, 83-naphthoflavone pretreatment of fish resulted in a significant 40% increase in the level of P-450 hemoprotein.
It is noteworthy that the X,flax for the carbon monoxide complex of reduced cytochrome P-450 was at 449 nm in all preparations examined (Fig. 6).

Discussion
The present study has demonstrated that PCBs and PBBs are capable of stimulating hepatic microsomal monooxygenation reactions in rainbow trout following a single intraperitoneal injection. In general, Aroclor 1254 and Aroclor 1242 appeared to be more potent than FireMaster BP-6; however the latter compound was much more effective at in-Environmental Health Perspectives ducing ethoxyresorufin-O-deethylation.
Notably Aroclor 1242 treatment resulted in monooxygenase activities, attaining a maximum at about 4 days, while in Aroclor 1254-treated fish maximal stimulation was not observed until 7 days. FireMaster BP-6 increased AHH and ethoxyresorufin-O-deethylation by 4 days after injection, however ethoxycoumarin-O-deethylation did not reach a maximum until 7 days after injection. This has also been observed in rats treated with Fire-Master BP-6 (14).
The inducing properties of the polyhalogenated biphenyls appear to be expressed differently in fish than in rodents. The polyhalogenated biphenyls failed to increase liver/body weight ratios or yield of microsomal protein; furthermore increases in ethylmorphine-N-demethylase were not apparent. These responses are typical of polyhalogenated biphenyl induction in rodents (10)(11)(12)(13)(14)(15)43) and are characteristic of phenobarbital-type (cytochrome P450) induction. PCB and PBB treatment of rainbow trout were found to stimulate AHH, ethoxycoumarin-O-deethylation and ethoxyresorufin-O-deethylation. AHH and ethoxycoumarin-Odeethylation are induced by both phenobarbital and 3-methylcholanthrene in rodents; however ethoxyresorufin-O-deethylation is specifically inducible by the polycyclic aromatic hydrocarbon type of inducing agents [cytochrome P1450] (38).
Because of the ability of commercial polyhalogenated biphenyls to elicit both phenobarbital-like and 3-methylcholanthrene-like induction, the term 'mixed' inducer has frequently been used. It has been demonstrated that cytochrome P1-450 or cytochrome P-450 induction by PCBs is determined by the substitution pattern of the halogen atoms (43)(44)(45). For example 2,2' ,4,4' ,5 ,5'-hexachlorobiphenyl was found to induce a cytochrome P-450, while 3,4,3',4'-tetrachlorobiphenyl specifically induced cytochrome P-448. More recently (46,47) it has been suggested that a similar situation exists with the PBBs. These workers have demonstrated that 2,4,5,2',4',5'-hexabromobiphenyl (the major component of FireMaster BP-6) is a cytochrome P-450 inducer (phenobarbital type). By extrapolation from data obtained using the PCBs it is logical to assume that certain PBB isomers may induce cytochrome P1-450. However, some evidence exists for the hypothesis that the 'mixed' properties of PBB-induced microsomal preparations are not due to the presence of cytochrome P1-450 (48).
In contrast to the apparent "mixed" patterns of stimulation of monooxygenation in rodents due to polyhalogenated biphenyls, rainbow trout seem incapable of responding to phenobarbital-type (cytochrome P-450) induction.
The large increases (up to 25-30-fold) in monooxygenase activity observed after PCB or PBB treatment of fish cannot be explained in terms of an increase in total hemoprotein P-450 concentrations, since the levels of this (these) enzyme(s) were increased only by about 10-20%. Hence it is possible that a novel enzyme with different substrate specificity is induced by the PBBs and PCBs in rainbow trout. However, no changes in the wavelength of the absorption maximum of the CO complex of ferrocytochrome P-450 were seen, nor were the ratios of the 450 and 430 nm absorption maxima of the ethylisocyanide complex of ferrocytochrome P-450 significantly altered. In rodents, the 455-530 peak ratio increases considerably after treatment with PCBs or PBBs (9,43,48).
Further studies utilizing specific inhibitors of cytochrome P1-450 (a-naphthoflavone) and cytochrome P-450 (metyrapone) demonstrated no distinct differences in the pattern of inhibition of monooxygenation in PCB, PBB or control hepatic microsomes obtained from rainbow trout (Elcombe, unpublished data).
In conclusion, it is apparent that PCBs and PBBs are potent and persistent inducers of certain monooxygenase reactions in the rainbow trout. These compounds, which show a "'mixed" type of induction in rodents, only elicit a polycyclic aromatic hydrocarbon type of stimulation in the rainbow trout. However, unlike the mammalian situation, this stimulation does not appear to be accompanied by the synthesis of cytochrome P1-450.