Mixed Function Oxygenase Induction Research Articles

Use of an mfo‐directed toxicity identification evaluation to isolate and characterize bioactive impurities from a lampricide formulation

AbstractRecently, a field formulation of the lampricide containing 3‐trifluoromethyl‐4‐nitrophenol (TFM) was identified as a potent inducer of mixed‐function oxygenase (MFO) detoxification enzymes in fish. It was further shown that induction was not associated with primary formulation ingredients. Using a toxicity identification evaluation (TIE) approach based on rainbow trout hepatic MFO activity, the TFM field formulation was investigated to isolate the compound(s) responsible for induction. Solid phase extraction and reverse‐phase high‐pressure liquid chromatography (HPLC) isolated activity in two distinct fractions, which were profiled by gas chromatography–high‐resolution mass spectrometry. The major constituents in both fractions were confirmed by synthesis as nitro‐, trifluoromethyl‐, and/or chloro‐substituted diphenyl ethers. However, fish exposures to the pure compounds failed to cause MFO induction. After further fractionations by HPLC, induction was determined in three new subfractions. Confident identification of a chloro‐nitro‐trifluoromethyl‐substituted dibenzo‐p‐dioxin has been made in two of these fractions. Although the specific chemicals responsible for induction have not been confirmed, a suite of impurities including chloro‐, and/or nitro‐, and/or trifluoromethyl‐substituted phenols, diphenyl ethers, and dibenzo‐p‐dioxins have been identified in the formulation. It is likely that these materials originate during industrial synthesis of TFM. These findings suggest that additional structurally related impurities are also present in this formulation.

Identification of Lampricide Formulations as a Potent Inducer of MFO Activity in Fish

White sucker caged in a Lake Superior tributary during a lampricide application showed marked induction of hepatic mixed function oxygenase (MFO) enzymes. Laboratory trials (0.5-5.8 mg L −1, 24 h exposure) with rainbow trout confirmed that field formulations of lampricides induce MFO activity. When the two primary components of the lampricide formulation were tested separately, MFO induction was associated with the 3-trifluoromethyl-4-nitrophenol (TFM) formulation, and not the 2’,5-dichloro-4’-nitrosalicylanilide (Bayer 73) component of the field application. Bioassays confirmed that reverse phase HPLC successfully separated the inducing component(s) of the TFM formulation. Induction was not associated with the HPLC fraction of the formulation containing TFM, which was corroborated by subsequent exposures of fish to purified TFM. These biochemical effects of lampricide exposure appear to be related to the presence of an unidentified contaminant in the field formulation. The significance of the MFO induction in terms of growth, reproduction, and survival is unknown, but exposure was associated with a transient decrease in circulating levels of steroid hormones.

Response of hepatic mfo activity and plasma sex steroids to secondary treatment of bleached kraft pulp mill effluent and mill shutdown

AbstractThe discharge of bleached kraft mill effluent (BKME) into Jackfish Bay, Lake Superior, Canada, has been associated with a number of changes in the physiology and whole organism responses of four fish species. Current studies have been following physiological indicators of BKME impact for evidence of improvement after the installation of a secondary treatment system. Secondary treatment has not been successful in eliminating BKME impacts on hepatic mixed‐function oxygenase (MFO) activity in white sucker (Catostomus commersoni) because fish collected from Jackfish Bay after initiation of secondary treatment exhibited similar MFO activity as recorded in samples collected during two previous years. Hepatic MFO activity was also induced in long‐nose sucker (Catostomus catostomus), lake whitefish (Coregonus clupeaformis), and lake trout (Salvelinus namaycush) after secondary treatment. However, samples collected two weeks after a planned mill maintenance shutdown showed no MFO induction in long‐nose sucker, reduced MFO activity in white sucker, and a reduced impact zone for MFO induction in lake whitefish. A reduction in circulating levels of gonadal sex steroids has also been recorded in fish exposed to primary‐treated BKME in Jackfish Bay. In contrast, neither secondary treatment nor mill shutdown was sucessful in eliminating impacts of BKME exposur on levels of testosterone and 17β‐estradiol in female white sucker and long‐nose sucker. The short duration of MFO induction after shutdown and the peristence of steroid reductions suggest that (a) secondary treatment has not been successful in removing “MFO‐active” compounds from BKME, (b) induction is not related to sediment contamination with persistent compounds, (c) the inducing agents are rapidly cleared by fish, and (d) effects on steroids may not be directly related to MFO induction.

Reproductive Dysfunction and MFO Activity in Three Species of Fish Exposed to Bleached Kraft Mill Effluent at Jackfish Bay, Lake Superior

Abstract Our recent studies have demonstrated reproductive dysfunction in white sucker (Catostomus commersoni), longnose sucker (C. catostomus) and lake whitefish (Coregonus clupeaformis) populations exposed to bleached kraft mill effluent (BKME). Although all three species show elevated levels of hepatic mixed function oxygenase (MFO) activity and depressed circulating steroid levels, we have been unable to provide clear evidence of whether these two events are directly linked to whole organism changes. Although depressed steroid levels appear to be linked to delayed sexual maturity, changes in fecundity and reduced secondary sexual characteristics in white sucker and lake whitefish, longnose sucker show no impacts of reduced steroid levels on reproductive performance. Installation of secondary treatment at this pulp mill did not alleviate the steroidal dysfunction or MFO induction. However, samples collected after a two week maintenance shutdown showed a return to reference levels of MFO activity in both sexes and of steroid levels in male fish. The relationship between elevated MFO activity and depressed steroid levels is unclear, but detailed experiments suggest that the two phenomena are not directly linked. White sucker show depressed steroid production and impaired reproductive regulation independent of MFO activity.

Persistent effects of a single dose of Aroclor 1254 on cytochromes P450IA1 and IIB1 in mouse lung

The polychlorinated biphenyl mixture Aroclor 1254 has been shown to elicit prolonged biochemical responses in several rodent species, particularly induction of mixed function oxygenases in hepatic tissue. Lung is also of interest since a single dose of Aroclor 1254 has been demonstrated to have a tumor promoting effect, increasing the numbers of lung tumors in Swiss mice initiated with N-nitrosodimethylamine. To investigate the enzyme induction response in lung, male Swiss mice were given a single 100 or 500 mg/kg dose of Aroclor 1254 and euthanized at time intervals ranging from 48 hr to 30 weeks. Both cytochromes P450IA1 and IIB1 were followed by use of specific enzyme activities and Western immunoblotting. The IA1 isoform, as quantified by ethoxyresorufin- O-deethylase activity and immunoblotting with monoclonal antibody 1-7-1, was significantly elevated for 30 weeks after both doses. In contrast, benzyloxyresorufin- O-dealkylase activity (P450IIB1 specific), which is constitutively expressed in rodent lung, was unaffected by Aroclor treatment at the lower dose at early time points, but induced twofold at 30 weeks. At the higher dose, however, enzymatic activity was decreased to 50% of control values, an effect which persisted for 4 weeks postexposure. These changes were confirmed by Western immunoblotting utilizing monoclonal antibody 2-66-3. Concomitantly, content of individual PCB congeners in lungs and carcass was quantified by gas chromatography with electron capture dectection. One congener, 2,3,3′,4,4′-pentachlorobiphenyl, was selectively retained in lung compared to carcass. Lack of correlation between changes in lung content of PCBs and levels of the P450 isoforms suggested interactions between congeners in control of P450 induction and repression. These data confirm a prolonged P450 induction response in nonhepatic tissue following Aroclor exposure, and further suggest a bidirectional role for certain PCB congeners in the regulation of P450IA1 and P450IIB1 expression in lung tissue.

Use of mixed‐function oxygenases to monitor contaminant exposure in wildlife

AbstractThis overview examines the utility of mixed‐function oxygenase (MFO) enzymes as a bio‐effects monitor for wildlife (amphibians, reptiles, birds and mammals) in view of their widespread use as indicators of contaminant exposure in aquatic invertebrates and fish. Phylogenetic trends in MFO activity, toxicological implications of induction and the relationship between contaminant exposure and MFO activity are discussed. Field studies using avian embryos and hatchlings suggest that MFO induction has utility for documenting contaminant exposure; however, findings in adult birds and mammals are equivocal. Age, sex and season are sources of variation that require consideration when undertaking field trials. Further understanding of MFO inducibility among species and application of recently developed analytical techniques including quantification of specific cytochrome P‐450 isozymes are warranted.

The interplay between mixed function oxygenases and DNA alteration under PAH pollution

Our experience in DNA alteration assessment tells us that aquatic vertebrates, in general, and also some of the invertebrates, by living in polluted areas acquire altered DNA; DNA alterations being defined either as single-strand breaks, and/or alkali sensitive or single-strand specific nuclease-cleavable sites (sse). This can be shown by several methods: (i) Alkaline filter elution; 1 (ii) fluorescence analysis of DNA unwinding; 2 (iii) alkaline sucrose density gradient centrifugation; 3 (iv) alkaline unwinding analysis with hydroxyapatite differentiation; 4 (v) nucleoid sedimentation analysis; 5 (vi) supercoiled DNA relaxation detection by electrophoresis; 6 and (vii) measurement of DNA molecular weight distributions with the electron microscope. 7 The amount of sse per DNA mass can be a function of either an increased activity of DNA damaging agents or agents that inhibit DNA repair. Among DNA damaging agents are polycyclic aromatic hydrocarbons (PAH), that can cause sse by the ‘fast effect’ 8 or after induction upon uptake, through the action of mixed function oxygenase (MFO). Since MFO induction within certain limits is a function of PAH concentration measurement of this enzyme activity enables at least partial interpretation of the DNA alteration status, which can be a two-phase process.

Mixed-function oxygenases as biological monitors around petroleum hydrocarbon development sites: Potential for induction by diesel and other drilling mud base oils containing reduced levels of polycyclic aromatic hydrocarbons

The use of oil-based drilling muds has been discouraged in hydrocarbon exploration and production in the marine environment but these muds are presently being used to a considerable degree in the United Kingdom and Norwegian sectors of the North Sea. Field studies in the North Sea have demonstrated only localized impacts around individual drilling sites, 1,2 even including those sites where ‘toxic’ diesel oils were employed as base fluids in drilling muds. Yet the question of disposal of cuttings contaminated with oil from drilling muds remains somewhat controversial. The induction of mixed-function oxygenases (MFO) has been validated on a number of occasions in the field as a sensitive index of low level hydrocarbon exposure (reviewed by Payne), 3 including sites in the North Sea where diesel-based muds were used. 4 The present study demonstrates that any potential for induction by hydrocarbon contaminated cuttings will probably be reduced by substitution of low-aromatic base oils for diesel in drilling mud formulations.

Hydrocarbon metabolism and cortisol balance in oil-exposed ringed seals, Phoca hispida

1. Ringed seals were exposed experimentally to oil contamination, by feeding of a [ 14C]naphthalene marked crude oil in fish for up to 4 days at a rate of 5 ml/day. 2. Mixed function oxygenase (MFO) activity, measured as aryl hydrocarbon hydroxylase in liver and kidney, was found to be induced, in particular in kidney tissue where the activity increased 3-fold. 3. MFO induction correlated with a high degree of conversion of crude oil hydrocarbons to water-soluble metabolites. Most of the radioactivity was found in the polar fraction of plasma and urine. 4. Plasma cortisol levels were somewhat elevated by captive holding, and increased markedly after oil-exposure. Cortisol half-life decreased after oil exposure from 1 3 4 to 1 hr.

Further evidence of cyclic amp-mediated hypertrophy as a prerequisite of drug-specific enzyme induction

We have previously reported that the administration of phenobarbital, 3-methylcholanthrene or the polychlorinated biphenyl, Aroclor-1254, to rats resulted in an early and sequential increase in the activities of hepatic cAMP-dependent protein kinase(s), omithine decarboxylase and RNA polymerase I. It was suggested that this sequence of events was involved in both liver hypertrophy and the induction of microsomal mixed-function oxygenases. To further test this hypothesis, we investigated if mice unable to induce aryl hydrocarbon hydroxylase in response to 3-methylcholanthrene exhibited increases in these cAMP-mediated events. A single dose of 3-methylcholanthrene (150 mg/kg, i.p.) was administered to male C57B1/6J (aryl hydrocarbon responsive) and DBA/2J (aryl hydrocarbon nonresponsive) mice. In the C57B1/6J mice, the hepatic cAMP concentration increased to 165 per cent of control within 1 hr. Maximal increases in the activities of liver cAMP-dependent protein kinase(s) (160 per cent), ornithine decarboxylase (210 per cent) and RNA polymerase I (120 per cent) occurred at 2, 4 and 6 hr, respectively, in the responsive mice. There were no detectable increases in any of these parameters in the nonresponsive (DBA/2J) mice. Multiple doses of 3-methylcholanthrene (20 mg/kg, i.p., daily × 3) resulted in increases in hepatic aryl hydrocarbon hydroxylase (460 per cent) and liver weight/body weight ratios (118 per cent) in the responsive (C57B1/6J) mice killed at 5 days. There was no increase in either of these parameters in the nonresponsive (DBA/2J) mice. Both the responsive and nonresponsive mice responded similarly to a single parental dose of phenobarbital (100 mg/kg) with maximal increases in the activities of cAMP-dependent protein kinase(s) (150 per cent), ornithine decarboxylase (160 per cent) and RNA polymerase I (135 per cent) at 2, 4 and 8 hr respectively. Multiple doses of phenobarbital (100 mg/kg, i.p., daily × 3) resulted in increased ethylmorphine N-demethylase activity (160 per cent) and liver weight/body weight ratios (130 per cent) in both strains of mice at 5 days. These data provide further evidence of coupled cAMP-mediated hypertrophy and induction of mixed-function oxygenases in liver.

Activation of 3':5'-cyclic AMP-dependent protein kinase and induction of ornithine decarboxylase as early events in induction of mixed-function oxygenases.

The parenteral administration of a single dose of 3-methylcholanthrene to rats caused an increase in the liver of the concentration of 3', 5'-cAMP and of the activity of cAMP-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37). These events were followed by an increased activity of ornithine decarboxylase (L-ornithine carboxy-lase, EC 4.1.1.17), the enzyme that controls the biosynthesis of polyamines. Finally, the activity of benzo[a]pyrene hydroxylase, as well as the amount of cytochrome P-448, was increased. Similarly, after the administration of phenobarbital, there was first an increase in the cAMP concentration and in the activity of cAMP-dependent protein kinase, then the induction of ornithine decarboxylase, and finally, an enhanced activity of ethylmorphine N-demethylase and an increased content of cytochrome P-450. These data suggest that the drug-induced processes in liver that increase the activities of the oxidative, and presumably other, drug-metabolizing enzymes include the following sequence of events: (1) increase in cAMP concentration and/or activation of cAMP-dependent protein kinase; (2) induction of ornithine decarboxylase; and, (3) induction of drug-metabolizing enzymes.