A novel, endogenous inhibitor of 7-ethoxyresorufin-O-deethylase activity isolated from liver cytosolic fractions of bream (Abramis brama L.)

Abstract

The cytosolic supernatant of bream (Abramis brama L.) liver homogenates inhibits the 7-ethoxyresorufin-O-deethylase (EROD) activity of pike (Esox lucius) microsomal fractions. The inhibitor shows no activity against 7-ethoxycoumarin-O-deethylase and benzo(a)pyrene hydroxylase indicating a high isoenzyme specificity. The inhibiting component is a heat-sensitive substance (56°C for 5′) which is not self regenerating after subsequent cooling. It can be isolated from the cytosolic fraction using two combined steps of ion exchange chromatography. The purification factor is 500-fold with a recovery rate of 70%. SDS–PAGE of the purified fractions indicate that electrophoretic purity was not achieved. However, a prominent band at about 97 kDa was present in all fractions in a close intensity–activity relationship. The molecular weight of the native form of the purified protein was determined to be 175±35 kDa using gel filtration on a Sephacryl S 300 HR column. So far the inhibitor can be characterized as a protein. It shows strong tendencies to aggregate due to lipophilic interactions. These interactions can be repressed by the addition of 1% sodium cholate. The inhibitor has an optimum activity at 25°C and pH 8.0. The inhibitor does not correspond to any of the known cytosolic, endogenous inhibitors of EROD activities in fish, including proteases, cytosolic phosphatases, kinases and resorufin reductase (e.g. DT-diaphorase), although a non-dicoumarol (10 μM)-inhibited menadione oxidoreductase activity of up to 46.7±0.4 nmol/min per mg inhibitory protein was measured. Kinetic studies using Michaelis–Menten kinetics with purified inhibitor fractions prove a non-competitive mode of inhibition. As this kind of inhibitor is not described yet it is named CERODIP (cytosolic, EROD-inhibiting protein).

Introduction

In recent years many attempts have been made to establish bioindicator systems to measure the toxic effects of highly lipophilic xenobiotics in aquatic systems. One approach was to measure the activity of microsomal monooxygenase activities in aquatic species [2], [17], [18] using for example the 7-ethoxyresorufin-O-deethylase (EROD) activity located mainly in liver microsomes. This cytochrome P450 isoenzyme belongs to the cytochrome P4501A family [25] and plays an important role in the biotransformation of planar lipophilic xenobiotics in fish. Due to sterical incompatibilities the oxygenated products of the isoenzyme activities of this family are not conjugated with glucuronic acid by phase II biotransformation enzymes. This leads to the formation of toxic intermediates. These intermediates are suspected to cause necroses, cancer and other cell damages [29]. The activities of the fish cytochrome P4501A isoenzymes are highly inducible by 3-methylcholanthrene type inducers like for example benzo(a)pyrene, 2,3,7,8-tetrachlordibenzo-d-dioxin (TCDD) and β-naphtoflavone [32]. Other factors known to be involved in the modulation of the microsomal monooxygenases activities are ambient temperature, seasonal changes, age, sex and hormonal status. At the time little is known about possible regulation mechanisms of this enzyme activities by other endogenous factors [35].

Most of the reported inhibitors of cytochrome P450 activities are small, exogenous molecules [6], [13], [20], [21], [26], [33], [40]. Only a few endogenous inhibitors of cytochromes P450 activities have been reported in cytosolic fractions so far especially from mammals. These are DT-diaphorases (e.g. resorufin reductase), proteases, phospholipases, tyrosinase, kinases, phosphatases and antibodies of the IgG type, but also hormones like estrogen [12], [13], [16], [26], [27], [35], [41]. However, IgG type antibodies have not been reported for fish yet [9], [31]. In crustaceans a heat sensitive, high molecular inhibitor of 7-ethoxycoumarin-O-deethylase (ECOD) and benzo(a)pyrene hydroxylase (AHH) activity was identified. It inhibits fish microsomes in vitro [14]. Unfortunately, no further information is available concerning this type of inhibitor.

During our investigations on different cytochrome P450 activities in liver from rainbow trout (Oncorhynchus mykiss), derived from a fish farm in Berlin, a striking difference in the individual activity patterns of EROD, ECOD and AHH activity became obvious. Whilst the activity of ECOD and AHH were at the same level the EROD activity exhibited strong individual differences. This had to be due to internal factors as the individuals were from the same stock and age and kept under identical conditions. After examination of the different cell fractions an inhibitor of EROD activity was found in the cytosolic fraction of the liver homogenates.

A previous publication from our laboratory [1] reported the appearance of the inhibitor in cytosolic fractions in the fish species rainbow trout, bream, perch (Perca fluviatilis) and roach (Rutilus rutilus) caught in different lakes in the Berlin area. The inhibitory activity was found in almost all examined individuals, showing a seasonal variation with highest activities in the late autumn (November). In contrast the EROD activity was lowest at this time and vice versa in spring. The inhibitory activity was shown to be interspecific as for example bream liver cytosolic fractions also inhibit the EROD activity of perch, rainbow trout, pike, crayfish (Orconectes limosus) and marmoset monkey (Callithrix jacchus).

This study deals with the preliminary purification, characterization and kinetic properties of this inhibitor using bream cytosolic fractions. Pike (Esox lucius) liver microsomal fractions were used as source of EROD activity.