Mixed Function Oxidase Enzyme Research Articles

Clonidine effects on disposition of xenobiotics in rats: inhibited elimination of flow-limited but not extraction-limited agents.

1. The alpha 2-adrenoceptor agonist, clonidine, reduces the hepatobiliary clearance of the anionic dye, sulphobromophthalein (BSP) in rodents. We now compare the effects of clonidine on BSP elimination with its effects on disposition of compounds which are metabolized by hepatic microsomal mixed function oxidases. 2. BSP, 100 mg kg-1 was administered i.v. to rats at 4 h after s.c. saline or clonidine, 0.2 mg kg-1. Thirty min later, plasma BSP levels were 121.4 +/- 2.25 micrograms ml-1 in saline-treated rats, while in clonidine-treated rats they were 631.5 +/- 141.0 micrograms ml-1. Clonidine raised hepatic BSP levels from 256.0 +/- 28.9 micrograms g-1 tissue to 568.5 +/- 86.5 micrograms g-1. 3. Acute administration of clonidine (0.2 mg kg-1 s.c.) or repeated clonidine dosing (0.2 mg kg-1, s.c. twice daily for 10 days) did not affect the disposition of intravenously administered [14C]-antipyrine (15 mg kg-1). 4. Activities of the P450 mixed function oxidase enzymes, aniline hydroxylase and aminopyrine N-demethylase, were identical in liver microsomes from saline-treated rats and in microsomes from rats given single or multiple s.c. doses of clonidine (0.2 mg kg-1). 5. Addition of clonidine or other 2-substituted imidazoles at concentrations up to 2 microM did not affect the activities of aniline hydroxylase or of aminopyrine N-demethylase in suspensions of rat liver microsomes. Other substituted imidazoles, including cimetidine, clotrimazole and metronidazole, at concentrations of 0.2 microM or higher, inhibited the activities of these microsomal enzymes. 6. Clonidine slowed BSP elimination, which is probably hepatic blood flow-limited, but not the extraction-limited elimination of antipyrine, which is metabolized by hepatic microsomal enzymes.

2,4-Dichlorophenol and MCPA in a V79 Test

Two industrial chemicals, 2,4-dichlorophenol and 4-chloro-2-methylphenoxyacetic acid (MCPA), which have no toxic effects on the Chinese hamster cell line V79 alone, were tested for toxicity and mutagenicity in a cell-mediated test, where mixed-function oxidase (MFO) enzymes are active in the metabolism of xenobiotics. For 2,4-dichlorophenol, a dose-dependent toxicity as well as a slight mutagenicity could be shown when oxygenation enzymes were present. A similar degree of toxicity in a plant test system (the Allium test) indicates a similar risk of damage from exposure to dichlorophenol treatments in both these systems. MCPA did not induce any toxic or mutagenic effects at the concentrations tested. These results were not in agreement with previous results in plant material, where MCPA was clearly toxic at relatively low doses. However, since chlorophenols have been found in plants sprayed with phenoxyacetic acids, further investigations should be performed concerning potential risk to human beings.

Response of the mixed-function oxidase system to toxicant dose, food and acclimation temperature in the bluegill sunfish

The degree of induction of hepatic mixed function oxidase (MFO) enzymes in fish is modulated by environmental conditions. This study was designed to investigate the influence of water temperature, presence or absence of food, and exposure to benzo(a]pyrene (BaP) on the inductive response of bluegill sunfish ( Lepomis macrochirus). The results show an increase in 7-ethoxyresorufin O-deethylase (EROD) activity with an increase in acclimation temperature and dose. This activity appears to be associated with a very small fraction of the total cytochrome P-450 induced. Major changes were observed in the 53- and 57-kilodaltons (kDa) electrophoretic bands.

Seasonal variation of hepatic mixed function oxidases in winter flounder (Pseudopleuronectes americanus)

Abstract Indices of hepatic mixed function oxidase (MFO) activity in winter flounder ( Pseudopleuronectes americanus ) were measured at approximately monthly intervals from November 1983 to October 1985. Benzo[a]pyrene hydroxylase and ethoxyresorufin O -deethylase activities and cytochromes P450 and b 5 were generally, but not significantly, higher in males than in females. The MFO activity varied seasonally, reaching a maximum during or shortly after spawning. Variation in MFO enzyme activity between sexes was never greater than 2-fold at any time, and within sex, no greater than 6-fold during the seasonal cycle. This variability is less than that caused by exposure to environmentally realistic levels of some pollutants; measurements of MFO activity in this species might therefore be used to indicate environmental contamination.

Hydrocarbons and Complaints About Fish Quality in the Mackenzie River, Northwest Territories, Canada

Abstract The Mackenzie River in northwestern Canada is the largest North American source of freshwater to the Arctic Ocean There are continuous discharges of petroleum into the river from an oil field at Norman Wells both from refinery and natural seepage sources. Recently oil production was expanded using several artificial islands constructed in the river Coincident with the expansion native DENE fishermen in downstream communities complained that the quality of fish deteriorated. Specifically, the liver of burbot (Lota lota) was reported to have become small and dark in colour, and our investigation of that complaint is the subject of this report. Examination of the burbot revealed that the liver condition was associated with a low content of fat The question posed was whether this quality problem could be related to petroleum inputs from Norman Wells. Residue analyses of the fish showed low, but consistent, contamination with low-boiling aromatic hydrocarbons, with fish taken in winter more highly contaminated than fish taken in summer. The low levels in summer fish were found in fish from many freshwater locations with no obvious connection to a petroleum source This may reflect a broad atmospheric dispersal of these materials. Burbot from the lower Mackenzie River did not have elevated liver mixed-function oxidase enzyme activities relative to a reference population from Lake Winnipeg Experimental treatments with oil induced these enzyme activities, and so fish seem unlikely to have been affected by the enzyme inducing components of petroleum. Experimental starvation of burbot in the laboratory resulted in a loss of liver fats and in development of apparently the same liver condition The condition of the fish seems more likely to be of natural origin through factors related to nutrition or parasitism, although pollution cannot be ruled out definitively. Unexpectedly high concentrations of toxaphene (up to 5000 ng/g wet weight) and other organochlorines were found in burbot livers

Effects of polychlorinated biphenyls on mourning dove reproduction and circulating progesterone levels.

PCBs acquired in sublethal doses increase activation of the liver's microsomal action and hepatic mixed function oxidase (MFO) enzymes. Physiological effects of increase in the MFO system may delay, if not eliminate, avian reproductive behavior and nesting success. No evidence is yet available to clearly link PCBs to lowered levels of circulating steroids in birds. However, data concerning the effects of PCBs on avian reproduction are abundant. Increased degradation of circulating steroids might alter essential hormone dynamics associated with nesting behavior and ovulation. Alternatively, normal feedback regulation by the pituitary-gonadal axis might lead to a full compensatory response to increased MFO such that hormone profiles remain unaltered, suggesting alternative modes of action for PCBs in reproductive disorders. Therefore, the authors investigated the effect of dietary PCBs on circulating progesterone levels prior to and during egg laying in wild-trapped mourning doves (Zenaida macroura).

Effects of plane of nutrition and phenobarbital during the pre-mating period on reproduction in ewes fed differentially during the summer and mated in the fall.

An increased plane of nutrition of ewes before mating (flushing) has been associated with an increased number of lambs per ewe. The following study was done to investigate a possible mechanism for the flushing effect. During the summer, 98 Hampshire and Suffolk ewes grazed pastures with 50 of the ewes supplemented with .45 kg of corn grain X head-1 X d-1. For approximately 12 d before fall mating, each ewe received daily either .45 kg of a corn grain-soybean oil meal mixture, an oral dose of 1 g phenobarbital or no treatment. Liver biopsies were collected from 23 ewes, representing all treatment combinations, 3 d after initiation of pre-mating treatment for determination of concentration of hepatic mixed function oxidase enzymes (MFO). Summer supplementation vs no summer supplementation resulted in ewes with heavier (P less than .05) body weights near mating (75.6 vs 71.1 kg), higher (P less than .01) mating condition scores (3.2 vs 2.9), a greater (P less than .01) number of ovulations (1.99 vs 1.74), but little difference (P greater than .10) in number of lambs born per ewe lambing. Pre-mating treatment with phenobarbital or grain mix resulted in a greater number of ovulations (P less than .01, P less than .10, respectively) than no pre-mating treatment (2.14, 1.85, 1.60; respectively). Ewes given phenobarbital had more (P less than .10) lambs per ewe than ewes given no treatment (1.71 vs 1.40). Both phenobarbital and grain mix resulted in a significant increase in concentration of MFO compared with no treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Effects of Scotian shelf natural gas condensate on the mummichog

The 96h LC 50 of the water soluble fraction (WSF) of natural gas condensate from the Sable Is., N.S. area to Fundulus heteroclitus was between 4 and 5 μg · ml −1 naphthalene equivalents (initial concentration). Behavioural responses in the fish to sub-lethal concentrations of WSF were similar to those recorded for crude and fuel oils. Induction of the hepatic mixed function oxidase enzyme enthoxyresorufin O-de-ethylase (EROD) in sexually immature fish occurred at WSF concentrations below the LC 50, but during the prespawning period no induction occurred. EROD activity in unexposed fish showed seasonal variation consistent with differences in its inducibility.

Induction of mixed-function oxidase activity in mouse lymphoid tissues by polycyclic aromatic hydrocarbons.

Polycyclic aromatic hydrocarbon (PAH) exposure can cause mixed-function oxidase (MFO) enzyme induction in certain tissues of various organisms. Measurement of such induction might serve as a useful bioindicator of human exposure to PAHs, provided readily obtainable human tissues can be utilized for such measurements. We have investigated the MFO activity in various lymphoid tissues of the C3H mouse as a model system and have studied the effect of systemic PAH treatment on such enzyme activity. An MFO enzyme assay was used to measure the activity of 7-ethoxyresorufin deethylase, an enzyme activity that may be specific for the cytochrome P-448 subset of MFO enzymes (those enzymes that are induced in cells or tissues following PAH administration). Intraperitoneal injection of mice with 180 mg/kg (4.6 mg) benzo[a]pyrene (BaP) or 160 mg/kg (4.0 mg) 3-methylcholanthrene (MC) produced a significant induction in MFO activity in mouse spleen S9 fractions 48 h after the injection. Induction ratios (induced activity/control activity) between 4 and 5 were seen with BaP; MC produced induction ratios of 2.5-3.0. Enzyme activity was not induced in the spleen within 16 h following BaP or MC administration. Other experiments indicated that MFO activity could be induced in thymus cells 48 h after either BaP or MC treatment. Treatment with BaP or MC did produce significant enzyme induction in the liver and lung tissues from the animals both 16 and 48 h after chemical treatment.

Correlation between the induction of micronuclei in bone marrow by benzene exposure and the excretion of metabolites in urine of CD-1 mice

Male and female CD-1 mice received single oral doses of benzene (220, 440, and 880 mg/kg) and were pretreated with modifiers of the mixed-function oxidase enzyme activities. Urinary metabolites (MT) (0–24 and 24–48 hr) were quantified by high-performance liquid chromatography. The micronucleus test was performed at 30 h. The following pretreatments were used to correlate micronucleus formation and the excreted benzene MT: 3-Methylcholanthrene and β-naphthoflavone led to a marked increase in micronuclei (MN) and MT, whereas phenobarbital caused a slight increase, and SKF-525A had no effect. MN and MT were decreased when benzene was administered by the ip route or toluene was given simultaneously. Females had a lower number of MN and excreted more unconjugated phenol than did males. Muconic acid, hydroquinone, and phenol glucuronide and MN correlated well. They were dependent on both the dose and route of administration of benzene, being most inducible by P-448 inducers, in males more than females. The administration of hydroquinone induced MN, but phenol or catechol (200, 250, and 150 mg/kg, po, respectively) did not, and none of these compounds yielded trans,trans-muconic acid, a benzene MT in urine. This study establishes that benzene myeloclastogenicity is a function of its metabolism and that quantification of urinary metabolites could provide reliable correlates of this effect in vivo.

Free radical involvement in long wavelength UV light activation of nitrosamines to mutagens

Nitrosamines are carcinogenic and mutagenic only after metabolic activation via endoplasmic reticulum bound mixed function oxidase enzyme systems. Rencently a new photochemical process has been discovered by which nitrosamines are converted into unknown mutagenic compounds by irradiation with long wavelength UV light (> 335 nm) in the presence of phosphate ion at neutral pH. The mutagenic activity is detected by Ames Salmonella Typhimurium strain TA100 in the absence of rat liver microsomes. We have shown that mutagen production with nitrosomorpholine is inhibited in the presence of light by various spin trapping agents (N-t-butyl-phenylnitrone, etc.). Concurrent with this inhibition a stable free radical signal has been detected whose kinetics of formation is similar to the time course of mutagen formation during irradiation in the absence of spin trap. The free radical signal is formed only when phosphate or similar ions are present in the reaction mixture. Monomethylphosphate and dimethylphosphate can substitute for phosphate ion but with small ESR signals and mutagen formation. Trimethylphosphate gives a weak, time independent ESR signal and does not cause mutagen formation. The ESR splitting constants (aN and aH) for signals generated with each of the different phosphate species show differences which suggest that these ions may be components of some intermediate free radical species that is involved in stable mutagen formation. Arsenate ion inhibits mutagen formation in the presence of phosphate but is able in the absence of phosphate to form a ESR signal similar to that observed with phosphate ion.

Correlation between induction of xenobiotic metabolism and DNA damage from chemical carcinogens in the chick embryo in vivo.

Chick embryo liver and lung have high levels of basal mixed-function oxidase (MFO) enzyme activities during embryonic development. These activities are differentially inducible by 3,4,3',4'-tetrachlorobiphenyl (TCB) and phenobarbital (PB) in liver but are not inducible in lung. Nucleated chick embryo erythrocytes have no detectable basal or inducible MFO activities. Following induction by TCB or PB, embryos were exposed to the direct-acting carcinogen methyl methane-sulfonate (MMS) or the indirect-acting carcinogens aflatoxin B1 (AFB1) or 7,12-dimethylbenz[a]anthracene (DMBA). DNA damage was measured in liver, lung and blood by the DNA alkaline elution technique. Maximum DNA damage by MMS in liver, lung and blood and by AFB1 and DMBA in liver and lung occurred approximately 3 h after exposure. DNA damage was rapidly repaired in liver and lung, but was not repaired in blood. AFB1 and DMBA caused no detectable DNA damage in blood, even following PB or TCB induction. MMS-induced DNA damage was unaffected by induction in all groups with the exception that PB decreased DNA damage in liver at the low dose of MMS. AFB1- and DMBA-induced DNA damage was increased in liver and decreased in lung following induction by PB and TCB in 14-day embryos. In contrast, AFB1-induced DNA damage was increased by PB and decreased by TCB in 7-day embryonic liver. These results suggest that MFO induction can increase or decrease DNA damage in vivo, but that the direction and magnitude of change in DNA damage can vary depending on the inducer, carcinogen, tissue and the developmental age being investigated.

Formation and identification of naphthoquinone glutathione conjugates following microsomal metabolism of 1-naphthol.

Previous studies have indicated that 1-naphthol is metabolised by the cytochrome P-450 mixed function oxidase enzyme system to form a reactive species capable of covalently binding to microsomal protein (1,2). Furthermore, Hesse and Mezger (1) suggested the involvement of quinones and/or semiquinones in 1-naphthol-dependent covalent binding. In more recent studies, 1,4-naphthoquinone formed from 1-naphthol has been directly measured with both rat liver microsomes (2,3) and a reconstituted cytochrome P-450 enzyme system (4). From this, it was hypothesised that the toxicity of 1-naphthol, previously reported in isolated hepatocytes (5), may be mediated by naphthoquinone metabolites of l-naphthol.

Depression of mouse liver microsomal mixed function oxidase enzymes by adriamycin

Levels of cytochrome P-450 and cytochrome b 5, and activities of NADPH cytochrome c reductase and 7-ethoxycoumarin O-deethylase, were found to be significantly decreased in hepatic microsomes prepared from mice killed 24 h after administration of a single intraperitoneal (i.p.) dose of adriamycin (ADR, 5 mg/kg). In contrast, both ascorbate-induced lipid peroxidation and conjugated dienes were increased in the same preparations. In vitro addition of ADR (5 μg/ml) to hepatic microsomal preparations (1 mg/ml protein) from the control mice also led to a substantial decrease in the mixed function oxidase (MFO) enzymes. A characteristic spectral change with an absorption peak at 408 nm and trough at 422 nm was associated with this in vitro interaction. It is suggested that the loss of cytochrome P-450 and related MFO enzymes due to ADR treatment is related to the generation of free radicals and subsequent lipid peroxidalion in the liver.

Dietary fat—A requirement for induction of mixed-function oxidase activities in starved-refed rats

Male rats were starved 0–48 hr, and then refed diets containing 0% (F.F.) to 20% corn oil (C.O.) lab chow or 20% coconut oil (C.C.O.) for 1–4 days. Some received phenobarbital sodium (80 mg/kg, i.p. daily) for 1–3 days prior to decapitation. Five cytochrome P-450-dependent indicators were assayed as measures of altered hepatic microsomal function: ethylmorphine N-demethylase (EMDM), N-nitrosodimethylamine (DMN)- Ndemethylase, aniline hydroxylase (AH), benzo[ a]pyrene hydroxylase (AHH) and CO-difference spectra (P-450). Increasing dietary corn oil (0, 0.5, 10, 20%) in control rats resulted in a progressive increase in the activities of these five enzymes. Dietary fat influenced phenobarbital (Pb) inducibility of all mixed-function oxidase (MFO) enzymes measured except AHH. Pb induced the remaining enzymes only 11–22% in animals fed fat-free diet as compared to 119–246% in animals fed coconut oil and corn oil. Rats fed fat-free diet for 21 days without prior food deprivation and administered Pb had 79% more EMDM, 34% more AH and 120% more P-450 than non-induced controls, whereas rats fed 20% corn oil diet had 227% more EMDM, 143% more AH and 128% more P-450. A requirement of dietary fat for induction of MFO by Pb was demonstrated by these starvationrefeeding experiments. Coupled with data recovered from the 21-day studies, these experiments suggest that a compensatory mechanism may be operative during chronic feeding of the fat-free diet to partially return inducibility to the drug-metabolizing system.

Hepatocarcinogenicity of Benzo[<italic>a</italic>]pyrene to Rainbow Trout by Dietary Exposure and Intraperitoneal Injection<xref ref-type="fn" rid="FN1">2</xref><xref ref-type="fn" rid="FN2">3</xref>

The influence of benzo[a]pyrene [(BP) CAS: 50-32-8] on the induction of certain enzymes within the hepatic mixed-function oxidase (MFO) system and its potential carcinogenicity were examined in rainbow trout (Salmo gairdneri). Nine-week feeding trials were performed with 500 and 1,000 ppm BP to determine trout tolerance to BP. Levels of MFO enzymes, including ethoxyresorufin-O-deethylase (EROD), ethoxycoumarin-O-deethylase (ECOD), benzo[a]pyrene monooxygenase (BPMO), and cytochrome P450 were measured during this time. An 18-month feeding trial of a 1,000-ppm BP dose was initiated in duplicate groups of 100 fingerling rainbow trout. Samples of trout were killed at 6, 12, and 18 months for gross and histologic examination of the internal organs for neoplasms. A group of fifty 10-month-old rainbow trout were given 12 monthly ip injections of 1 mg BP in 0.4 ml propylene glycol (PG), and comparable controls were given PG injections only. The trout were held for an additional 6 months, killed at age 28 months, and examined as in the dietary study. Mean MFO enzyme levels of EROD, ECOD, BPMO, and cytochrome P450 were significantly (P less than .001) elevated, showing dose- and time-response relationships when compared to MFO enzyme levels in control fish. Twelve months after BP exposure was initiated, 15% of the BP-fed fish had histologically confirmed neoplasms of the liver. After 18 months the incidence increased to 25%. No evidence of neoplasia was observed in control fish. BP injected ip resulted in a 50% incidence of hepatocellular neoplasms and in a fibrosarcoma of the liver and papillary adenomas of the swim bladder in 1 fish. These results indicate that BP is a potent inducer of selected hepatic MFO enzymes and establish, for the first time, the hepatocarcinogenicity of BP in an aquatic species.

Toxicity of carbon disulfide in developing rats: LD50 values and effects on the hepatic mixed-function oxidase enzyme system

The 24-hr LD50 values of carbon disulfide (CS 2) were estimated in 1-, 5-, 10-, 20-, 30-, and 40-day-old rats. CS 2 was least toxic to 20-day-old rats (LD50 1545 mg/kg, ip) and most toxic to 1-day-old rats (LD50 583 mg/kg, ip). Twenty-four hours after administration of CS 2 (375 mg/kg, ip) to 1-, 5-, 10-, 20-, 30-, and 40-day-old rats, significant inhibition of cytochrome P-450 and aniline hydroxylation was observed in rats of all ages studied except the 1-day-old rats. Following incubation of hepatic microsomes isolated from 1-, 5-, 10-, 20-, 30-, and 40-day-old rats with CS 2, decreases in activity of the hepatic mixed-function oxidase enzyme system and/or concentration of cytochrome P-450 were observed when an NADPH-generating system was present during incubation. When hepatic microsomes isolated from rats of all ages studied were incubated with C 35S 2, 35S was covalently bound to microsomal protein in the presence of an NADPH-generating system. Also, more 35S than 14C was covalently bound to microsomal membranes after incubation of microsomes isolated from rats of all ages studied with C 35S 2 or 14CS 2 in the presence of an NADPH-generating system. The results of this research demonstrated the LD50 of CS 2, the effects of CS 2 on the hepatic mixed-function oxidase enzyme system, and that the conversion of CS 2 to a covalently binding sulfer-containing biotransformation product varied with age in developing rats.

Development of laser-excited, fluorometric assays for the monitoring of toxic chemical exposure

Abstract

The role of DNA-repair processes in N-nitrosopyrrolidine-induced mutagenesis.

The cytotoxic and mutagenic effects of increasing concentrations of N-nitrosopyrrolidine (NPYR) were studied using various DNA repair mutants of Escherichia coli together with rat-liver S9 activation system. Irrespective of which strain was used, the cytotoxic effects of NPYR were similar to those observed in the parent strain. Mutagenicity studies revealed that the uvrA- derivative was more mutable than its repair proficient parent. These observations suggest that NPYR reacts with DNA to generate bulky lesions, which although potentially mutagenic, do not contribute significantly to cell-killing. Subsequent experiments with the metabolic inhibitor SKF 525A revealed that this compound only partially inhibited the mutagenic activity of NPYR, suggesting that although hepatic mixed function oxidase enzymes may participate in NPYR activation other pathways of metabolism are also involved.

A correlation of induced multifunction oxidase activity to bond strengths in partially chlorinated hydrocarbons

Four xenobiotics, benzene, 1,2,4-trichlorobenzene, cyclohexane and γ-1,2,3,4,5,6-hexachlorocyclohexane were orally administered at 1 7 LD 50 dose levels to Sprague Dawley rats. Seventy-two hours after dosing, the specific activity of the hepatic MFO system of each animal was determined in vitro by measuring NADPH utilization when each of the four xenobiotics was added as the substrate. For each of the four dose types, the specific activity in vitro showed no dependence on substrate stereo-chemistry, but was inversely related to the C-H bond strength of the substrate. For each substrate, the specific activities of each of the four types of MFO preparations varied directly with the C-H bond strength of the inducer. This implies that the specific activity of the induced MFO enzyme system is adjusted to the C-H bond breaking requirement which leads to the hydroxylation of the xenobiotic. This hypothesis was further examined following in vivo exposure of rats to six polychlorinated derivatives of dibenzo-p-dioxin (PCDD). The specific activity of each MFO extract was determined by use of all other PCDD's as substrates. The dioxins showed a consistent ranking for both activity as substrate and as inducer but, unlike the monocyclic hydrocarbons, the two rankings were not a simple inverse of each other. By combining a series of in vivo and in vitro tests, a structure-activity-relationship can be developed to relate the mixed function oxidase (MFO) response to the physico-chemical properties of a xenobiotic. Following in vivo exposure to one of a series of hydrocarbons or their chlorinated