Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology
A novel, endogenous inhibitor of 7-ethoxyresorufin-O-deethylase activity isolated from liver cytosolic fractions of bream (Abramis brama L.)
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.
Section snippets
Chemicals
Bovine serum albumin, glucose-6-phosphate-dehydrogenase, glucose-6-phosphate, 7-ethoxyresorufin, resorufin, 7-ethoxycoumarin and umbelliferon were purchased from Boehringer (Mannheim, Germany). DEAE Sephacel, Sephacryl S 300 HR, Phenyl-Sepharose CL 4B and Mono Q HR 5/5 were obtained from Pharmacia (Uppsala, Sweden). All other chemicals were at least of analytical grade and purchased either from Sigma (USA) or from Merck (Darmstadt, Germany).
Animals
Adult bream and pike were caught by trawling either by
Localization of the inhibitor in the liver
The intracellular localization of the inhibitor was examined using differential centrifugation. Aliquots of liver homogenates were centrifuged at 10 000×g for 20 min to obtain the PMS consisting mainly of the cytosolic and microsomal proteins. An aliquot of the PMS was examined for inhibitory activity and used for Ki determination. The rest of the PMS was centrifuged at 150 000×g for 1 h to separate the cytosolic fraction (CF) from the microsomal fraction (MSF). The Ki of the CF was determined
Discussion
More than 60 inhibitors of cytochrome P450 activity have been reported during the last three decades. However, most of them are of small size and exogenous [6], [13], [20], [21], [33], [40]. About ten of them are endogenous substances, but not one of them seems to be specific for cytochrome P450 isoenzyme activities. However, specific endogenous inhibitors of the cytochrome P450 system would be of great physiological importance, as this enzyme system is of great pharmaceutical and
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