Mixed Function Oxidase Activity Research Articles

Interannual Mixed Function Oxidase (MFO) Activity in Winter Flounder (Pleuronectes americanus) from a Coal Tar Contaminated Estuary

7-Ethoxyresorufin-O-deethylase (EROD), benzo[a]pyrene hydroxylase (BaPH), and cytochromes P-450 (cyt-P450) and b5(cyt-b5) varied annually in winter flounder (Pleuronectes americanus) collected in August of 1987, 1988, and 1989 from a coal tar contaminated estuary (Sydney, Nova Scotia, Canada). For August 1989, with fish available from all estuary areas, these indices correlated strongly with a spatial (along estuary) gradient in PAH in bottom sediments (7.19 ± 6.59–191 ± 184 μg g dry weight−1). Mean EROD activities in flounder near the coal tar source were up to seven times those in other estuary areas and paralleled sediment PAH loadings; however, standard deviations were high. Correlations for all MFO indices and sediment PAH were obtained in female flounder (P < 0.01: EROD, cyt-b5, cyt-P450; P < 0.02: BaPH). For male flounder the trend was similar, but only cyt-P450 correlated with sediment PAH (P < 0.017). BaPH activity was highest near the coal tar source but was more variable and less sensitive to pollutant levels than EROD activity. Somatic indices in fish from Sydney estuary and St. George's Bay were similar. Winter flounder are vulnerable to PAH-induced MFO activities from coal tar contaminated sediments, but MFO induction does not occur equally in all fish; single-season or single-year data must be interpreted with caution.

Mixed-Function Oxidase and Glutathione S-Transferase Activities from Field-Collected Larval and Adult Tobacco Budworms, Heliothis-Virescens (F.)

Mixed-function oxidase and glutathione S-transferase activities were measured from tissue homogenates of larval and adult tobacco budworms that were collected from cotton fields in Texas and Louisiana. Mixed-function oxidase activity toward p-nitroanisole was detected in both developmental stages and, in six of the eight field collections tested, mean levels of activity were significantly higher than activity measured in parallel assays with insects from a laboratory-reared, insecticide-susceptible colony. However, the significance of this activity with respect to pyrethroid metabolism and resistance is unclear because variation in mixed-function oxidase activity among these strains was poorly correlated with susceptibility to cypermethrin. Glutathione S-transferase activities toward 3,4-dichloronitrobenzene and 1-chloro-2,4-dinitrobenzene were also detected in both larvae and adult insects, and mean levels varied among the field collections tested. Patterns of activity in tissues examined for both mixed-function oxidases and glutathione S-transferases were different between larvae and adults suggesting that the regulation and expression of these enzyme activities is dependent on developmental stage.

The role of metabolic enzymes in insecticide‐resistant colorado potato beetles

AbstractSeveral metabolic enzymes were assayed in susceptible and insecticide‐resistant (R‐mfo and R‐AchE) strains of the Colorado potato beetle (Leptinotarsa decemlineata Say). We compared the activities of esterases, glutathione transferases and mixed function oxidases, and measured in‐vivo metabolism of carbofuran to determine the contributions of metabolic factors to the observed resistance. There were no substantial differences between strains in α‐ or β‐naphthylacetate hydrolysis, or in glutathione transferase activity. Compared to the susceptible strain, the R‐mfo strain showed 4‐fold higher activity in both ethoxyresorufin‐O‐dealkylase, and pentoxyresorufin‐O‐dealkylase activity. The R‐AChE strain had a 1.5‐fold higher level of ethoxyresorufin‐O‐dealkylase activity. Correspondingly, the in‐vivo metabolism of carbofuran was highest in the R‐mfo strain, and the R‐AChE strain was slightly higher than the susceptible strain. These results are discussed in relation to other resistance mechanisms which we have examined in these strains.

Biomonitoring of pollution: the hepatic cytochrome P-450 enzyme system in the feral mouse Apodemus flavicollis as indicator

The aim of the present study was to find a suitable wild-rodent to be used as terrestrial bioindicator. The wild mice were trapped at three sites around Vienna and at a fourth location in the countryside. Apodemus flavicollis, the yellow-necked mouse, was the most frequently captured mouse at all four sites. For each sample hepatic microsomal protein content, cytochromes b5 and P450, and enzyme catalytic activities-ECOD, EROD, BROD and PROD were measured. Except for one male urban group no significant differences between rural and urban groups in ECOD activity were observed. Increased cytochrome b5 and P450 contents per mg of microsomal protein were found in one of the male and all the three female urban groups. However the alkoxyresorufin O-dealkylase activities were significantly increased in both sexes of the three city groups. These results suggest that induction of cytochrome P450IA and P450IIB families has occurred in urban populations, indicating a possible pollution with halogenated dioxins and furans, PCBs, PAHs etc. The data show that induction of hepatic mixed function oxidase (HMFO) activity in the feral mouse Apodemus flavicollis can be successfully used as a terrestrial bioindicator for pollution monitoring.

Effects of in vivo benzo(a)pyrene treatment on liver microsomal mixed-function oxidase activities of gilthead seabream ( Sparus aurata)

Benzo(a)pyrene [B(a)P] treatment of gilthead seabream, 25 mg/kg, i.p. for 5 consecutive days, did not cause any significant changes in ethylmorphine N-demethylase and aniline 4-hydroxylase activities of liver microsomes. The same treatment did not alter the liver microsomal cytochrome b5 content, NADH-cytochrome b5 reductase and NADPH-cytochrome P450 reductase activities. However, benzo(a)pyrene treatment caused a 2–3-fold increase in 7-ethoxyresorufin O-deethylase (7-EROD) activity of gilthead seabream liver microsomes. Although, upon treatment, total cytochrome P450 content of liver microsomes increased about 1.7-fold in 1990 fall, no such increase was observed in spring 1991. However, a new cytochrome P450 with an apparent M r of 58,000 was observed on SDS-PAGE of liver microsomes obtained from benzo(a)pyrene treated gilthead seabream. Besides, in vitro addition of 0.2 × 10 −6 M benzo(a)pyrene to the incubation mixture inhibited 7-ethoxyresorufin O-deethylase activity by 93%. Gilthead seabream liver microsomal 7-ethoxyresorufin O-deethylase activity was characterized with respect to substrate concentration, amount of enzyme, type of buffer used, incubation period and temperature.

In vivo effects of the anesthetic, benzocaine, on liver microsomal cytochrome P450 and mixed-function oxidase activities of gilthead seabream ( Sparus aurata)

Gilthead seabreams were exposed to benzocaine, 4-aminobenzoic acid ethyl ester, 57 mg/l in sea water for 3 min, daily, for 2 or 3 consecutive days. The fish were killed 20 hr after the last treatment. Benzocaine treatment for 2 or 3 days resulted in 57% and 67% inhibition of liver microsomal aniline 4-hydroxylase and ethylmorphine N-demethylase activities,respectively. The total cytochrome P450 content of fish liver microsomes was unaltered following the 2-day benzocaine treatment. However, additional 3 min benzocaine treatment on day 3 reduced cytochrome P450 level by 50%. Benzocaine produced type II difference spectra with rabbit liver microsomes. Difference spectra of fish liver microsomes elicited by benzocaine were complex. The position of peak and intensity were greatly influenced by the concentration of benzocaine.

Effects of m-xylene on rat nasal cytochrome P450 mixed function oxidase activities

The effect of m-xylene on the rat nasal cytochrome P450 (P450) mixed function oxidase system was analyzed in vitro utilizing microsomes isolated 2, 12, and 24 h following intraperitoneal administration of this solvent in vivo. For comparative purposes, pulmonary and hepatic activities were also measured. Benzyloxyresorufin O-deethylation (BROD) and ethoxyresorufin O-deethylation (EROD), catalytic activities linked with P450 isozymes IIB1 and IA1, respectively, were inhibited in nasal tissue at all times following m-xylene administration. Pulmonary tissue mimicked this m-xylene-dependent inhibition of BROD activity but did not display significant inhibition of EROD activity. In contrast, m-xylene caused a dramatic induction of both BROD and EROD activity in hepatic tissue. The metabolism of a third P450 substrate, cyclopentadienyl manganese tricarbonyl (CMT), was also analyzed. m-Xylene caused significant inhibition of CMT metabolism at all time points in both nasal and pulmonary microsomes but was without effect on hepatic microsomal metabolism of this compound. These data show an inhibitory effect of m-xylene on rat nasal and pulmonary but not hepatic cytochrome P450-dependent metabolism.

Effects of Dietary Fat and Protein Sources on Performance, Lipid Content and Mixed Function Oxidase in Liver, and Fat Deposition and Adipocyte Cellularity in Abdomen in Broiler Chickens.

Abdominal fat deposition and the cellularity, and lipid content and mixed function oxidase (MFO) activity in livers were studied in broiler chickens fed isoenergetic and isonitrogenous diets which were different in the fat and protein sources.Feeding corn oil (CO) or chicken oil (CH) tended to decrease abdominal fat content compared with feeding yellow grease (YG) at 56 days of age, although no significant differences were observed in general performance. In female chickens, the decrease in the abdominal fat pad was associated with decrease in the adipocyte volume. At 21 days of age, lipid and triglyceride contents in the liver of chickens fed CO or CH were numerically lower than those in YG-fed chicks, and hepatic MFO activity was elevated by feeding CO.Feeding high fat diet (HF) increased liver triglyceride content at 21 days and tended to increase abdominal fat content at 56 days of age. Feeding fish meal (FM) as a major protein source increased the abdominal fat content in comparison with feeding soybean meal+fish meal (SFM), irrespective of dietary fat content, at 21 days of age. In chickens fed low fat diet (LF) for 21 days, FM decreased lipid and triglyceride contents in the liver and the decrease was associated with an activation of the hepatic MFO activity.

Mixed-function oxidase-specific activity in wild and caged speckled sanddabs Citharichthys stigmaeus in Elkhorn Slough, Moss Landing Harbor and nearshore Monterey Bay, California.

The goal of this study was to characterize the spatial, seasonal and annual hepatic activities of mixed-function oxidase (MFO) in the speckled sanddab Citharichthys stigmaeus, the most common fish in the Moss Landing area. In addition, techniques to monitor MFO activities in caged speckled sanddabs were developed and tested. Once the relationship between MFO activities in caged and wild fish populations is determined, caged fish could be used to monitor potential hydrocarbon impacts at Moss Landing, or other marine sites. During each of the spatial, seasonal and annual sediment samplings conducted in 1985-1987 as part of a separate hydrocarbon variability study at Moss Landing, 12 wild speckled sanddabs were collected from Moss Landing Harbor, Elkhorn Slough and nearshore Monterey Bay sites. In addition, four locations were chosen for a 14-day field caged fish experiment. The caged fish experiments successfully demonstrated the feasibility of using caged sanddabs as indicators of hydrocarbon exposure. The major source of variability in hepatic aryl hydrocarbon hydroxylase activity in wild speckled sanddabs from the Moss Landing area is due to seasonal rather than site differences. Significant relationships between caged fish MFO response and sediment hydrocarbon concentrations were found. It is possible that caged fish could be used in place of costly sediment sampling and analysis, and provide a more direct method to assess biological impacts. Many of the caging techniques demonstrated in this study can easily be transferred to other benthic flatfish, and other marine and freshwater environments.

Liver Microsomal Mixed-Function Oxidases in Response to Dietary n-3 Fatty Acid Levels in Rats

Phenobarbital (PB)-induced activities of the liver microsomal mixed-function oxidase (MFO) system in response to dietary levels of n-3 fatty acids were investigated in rats. The MFO activities were assessed by the contents of cytochromes P-450 and b5, and the activities of NADPH-cytochrome c reductase, aminopyrine N-demethylase and aniline hydroxylase. A linoleic acid (C18:2, n-6; LA) diet (2.0 energy % as LA) and n-3 fatty acid diets (0.3, 1.0, 1.9 or 4.8 energy % as n-3 fatty acids) were fed to rats for 16 days. The activities of MFOs except that of NADPH-cytochrome c reductase increased, as compared with the LA diet, with increasing the dietary n-3 fatty acid level, concomitant with the increase in n-3 fatty acids and decrease in n-6 fatty acids in liver microsomal lipids. The dietary LA over 1.8 energy % up to 13.8% did not increase the activities of PB-induced MFOs. Consequently, dietary n-3 fatty acids further elevated the PB-induced MFO activities effectively. These results suggest a differential response of dietary n-6 and n-3 fatty acids to the PB-induced MFO activities.

Liver microsomal mixed-function oxidases in response to polyunsaturated/saturated and n-6/n-3 fatty acid ratios of dietary lipids in rats.

The effects of various polyunsaturated/saturated (P/S; 0.29-2.74) and n-6/n-3 (0.49-11.21) ratios of dietary fatty acids and of phenobarbital (PB) stimulation on the activity of liver microsomal mixed-function oxidase (MFO) system were studied in rats using a combination of palm oil, safflower oil and fish oil concentrate. When the n-6/n-3 ratio was kept constant (4.6-4.9) without induction by PB, the highest P/S ratio (2.74) tended to increase the basal PB-uninduced MFO activities; while the PB-induced MFO activities were elevated as the dietary P/S ratio increased. This definitely indicates the role of both n-6 and n-3 polyunsaturated fatty acids (PUFAs) in the elevation of MFO activities with and without PB stimulation. Whereas the n-6/n-3 ratio was varied while maintaining a constant P/S ratio (1.3), the PB-induced MFO activities were significantly elevated by decreasing the n-6/n-3 ratio although the MFO activities were not affected without PB stimulation. The MFO activities induced by PB were directly related to the n-3 PUFA content and inversely related to the n-6 PUFA content of the diets with n-6/n-3 ratios less than 4.55. These results obtained herein suggest that there are significant differences in the effects of n-6 and n-3 PUFAs on liver microsomal MFO activities, especially in relation to the induction of MFOs by PB.

Blood cholinesterase in rats fed an insect resistance apple clone containing a natural cholinesterase inhibitor

A crab apple clone (Malus brevipes 1021), highly resistant to the apple maggot, is being used in breeding programs developing commercial apple cultivars. This study has discovered that this crab apple contains a natural cholinesterase (ChE) inhibitor that caused a 17.5% in vitro inhibition of rat blood ChE activity. This crab apple also showed a relatively high total (titratable) acidity of 1.28%. The commercial, nonresistant, apple cultivar McIntosh was capable of causing a 7.9% inhibition of blood ChE in vitro. The total acidity in McIntosh was 0.45%. A 4-wk feeding study compared 2 groups of 5-wk-old Fischer 344 male rats fed diets containing 45% of either M. brevipes or McIntosh freeze-dried apples to a third (control) group of rats fed a semipurified diet. In vivo blood ChE activities were similar in all groups of rats, as well as hemoglobin, hematocrit, and red blood cell counts. The liver mixed-function oxidase activity through aminopyrine N-demethylase in the rats fed the apple diets was higher than the controls, but p-nitroanisole O-demethylase activity was induced only in the animals fed the maggot-resistant crab apple. Lowered growth with concomitant lowered food intake, in the otherwise healthy rats fed the maggot-resistant crab apple diet, was attributed to the less palatable, highly acidic fruit. This study indicates that the natural ChE inhibitor in the insect-resistant apple M. brevipes is apparently detoxified upon ingestion.

Induction of cytochrome P4504A by the peroxisome proliferator perfluoro- n-octanoic acid

The influence of a single dose of the peroxisome proliferator, perfluoro- n-octanoic acid (PFOA) on hepatic and renal mixed-function oxidase activities has been examined in rats. Peroxisome proliferation was confirmed by increases in peroxisomal palmitoyl-CoA oxidation and carnitine acetyl transferase activity, particularly in liver. The liver was also more susceptible than the kidney to PFOA-dependent induction of the 12-hydroxylation of lauric acid, suggesting induction of the CYP4A sub-family. This was further confirmed by Western blot analyses, wherein an anti-CYP4A1 antibody revealed a substantial PFOA-dependent induction of CYP4A1 in a pattern similar to that observed for the classical peroxisome proliferator, clofibrate. In addition, using a cDNA probe to CYP4A1 in Northern blot analysis, PFOA treatment resulted in a marked increase in the steady state level of CYP4A1 mRNA, again more extensively in liver than in kidney. Taken collectively, our data provide compelling evidence that PFOA, like other peroxisome proliferators, is also an inducer of the CYP4A sub-family.

Effect of Dietary Alpha-Linolenic, Eicosapentaenoic and Docosahexaenoic Acids on Liver Microsomal Mixed-Function Oxidases in Rats

This study was undertaken to compare the effects of three major dietary n-3 fatty acids, i.e. alpha-linolenic acid (C18:3, n-3; LN), eicosapentaenoic acid (C20:5, n-3; EPA) and docosahexaenoic acid (C22:6, n-3; DHA), on liver microsomal mixed-function oxidase activities with and without phenobarbital (PB) induction. Male Sprague-Dawley rats were fed a 5% lipid diet containing either LN, EPA or DHA as major constituent fatty acid (about 40%) of dietary lipids for 15 days. The fatty acid compositions of liver microsomal lipids of each n-3 fatty acid group were characteristically different, particularly in the n-3 fatty acid compositions, but were nearly the same with and without PB treatment. Dietary LN, EPA and DHA did not affect the levels of PB-inducible cytochrome P-450 species nor the activities of aminopyrine N-demethylase and aniline hydroxylase. However, the PB-uninduced constitutive levels of cytochromes P-450 increased in the following order with a slight but significant difference between LN and DHA groups: LN group < EPA group < DHA group. Both the PB-uninduced activities of aminopyrine N-demethylase and aniline hydroxylase changed in a very similar manner to the constitutive levels of cytochromes P-450. These results suggest that the uninduced constitutive level of certain cytochrome P-450 species is modulated differentially by dietary LN, EPA and DHA, whereas dietary LN, EPA and DHA do not affect the PB-inducible levels of cytochrome P-450 species.

Hepatic metallothionein level and mixed function oxidase activity in fingerling rainbow trout (Oncorhynchus mykiss) after acute exposure to pulp and paper mill effluents

Hepatic metallothionein (MT) levels and mixed function oxidase (MFO) activity (7-ethoxyresorufin-o-deethylase or EROD) were measured in fingerling rainbow trout (Oncorhynchus mykiss) exposed to sublethal concentrations of 12 pulp and paper effluents, after completion of 96 h static acute lethality assays. Barring one primary-treated effluent where MFO levels were significantly depressed and two secondary-treated effluents where no significant MFO induction were observed, all other effluents triggered significant induction of MT and EROD, regardless of mill process/treatment or of effluent lethality and chemical characteristics. MT and EROD inductions were significant, however, at higher concentrations for secondary-treated effluents than for primary-treated ones. Lethal (96 h LC50s) to sublethal (MT and EROD lowest observable effect concentrations) ratios were variable and indicated that significant biochemical effects were present at effluent concentrations that were roughly 4–33 (MT) and 3–59 (EROD) times lower than the LC30. Enzyme induction ranged from 1.3 to 2.5-fold for MT and from 1.3 to 9.4-fold for EROD compared to controls. Limited chemical data available suggest that there were indeed classes of compounds present capable of inducing MT or EROD. Observed patterns of MT/MFO responses also suggest that contaminant interactions may have interfered with induction for some of the effluents studied. Refinements of this combined (sub)lethal bioassay procedure are envisaged to determine whether it can provide an efficient means of detecting hazardous chemicals in industrial wastewaters.

Effect of 4-(diethylamino)benzaldehyde on ethanol metabolism in mice.

The compound 4-(diethylamino)benzaldehyde (DEAB) is a potent inhibitor of cytosolic (class 1) aldehyde dehydrogenase (ALDH) in vitro and can overcome cyclophosphamide resistance in murine leukemia cells characterized by their high content of ALDH. In this study, we examined the in vivo effect of DEAB in mice on ethanol metabolism and on antipyrine clearance as a measure of the microsomal mixed function oxidase activity. DEAB administered in doses of 50 and 100 mg/kg increased the blood acetaldehyde concentration and decreased the plasma acetate concentration in mice treated with ethanol. A pharmacokinetic approach demonstrated that DEAB in doses of 50 and 100 mg/kg inhibited the fraction of ethanol converted to acetate by 32.5 and 67.5%, respectively. This inhibition was comparable with that produced by disulfiram. DEAB produced optimal inhibition of ALDH 10-15 min after administration. DEAB did not change the half-life or the total clearance of antipyrine. We conclude that DEAB is a potent inhibitor of ALDH in vivo and has no effect on the mixed function oxidase activity as determined by antipyrine clearance.

Induction of hepatic drug metabolizing enzymes and interaction with carbon tetrachloride in rats after a single oral exposure to atrazine

A single oral dose (430 mg/kg) of atrazine, a widely employed s-triazine herbicide, was administered to young male rats. There was a significant increase of the in vivo elimination ofhexobarbital and a significant induction of the activity of 7-pentoxyresorufin-O-dealkylase, while cytochrome P-450 content and other mixed function oxidase activities remained unaltered. The administration of carbon tetrachloride (CCI,) to atrazine pretreated rats did not substantially augment the impairment of drug metabolizing enzymes brought about by CCLi alone. Results suggest that atrazine behaves like a relatively weak inducer of phenobarbital-inducible families of cytochrome P-450.

The effects of inducing agents on cytochrome P450 and UDP-glucuronyltransferase activities in human HEPG2 hepatoma cells

Selective induction in vitro of cytochrome P450-dependent mixed-function oxidase (MFO) and UDP-glucuronyltransferase (GT) activities was observed in the human HepG2 hepatoma cell line. 1,2-Benzanthracene (BA) induced MFO O-dealkylation activities for ethoxyresorufin, methoxyresorufin and benzyloxyresorufin, whereas phenobarbitone (PB) selectively induced pentoxyresorufin O-dealkylation and rifampicin (RIF) selectively induced benzyloxyresorufin O-dealkylation. Antibody inhibition experiments indicated that ethoxyresorufin and methoxyresorufin O-dealkylations were catalysed mainly by the P450 1A subfamily in untreated and BA-induced HepG2 cells, that additional unidentified P450 forms were considerably involved in methoxyresorufin and benzyloxyresorufin O-dealkylations and that the P450 2B subfamily was partially responsible for pentoxyresorufin O-dealkylation in PB-induced cells. Bilirubin GT activity was induced by PB, BA, RIF and dexamethasone, but 1-napthol, morphine and testosterone GT activities were not induced by any of these treatments.

The effect of the cytochrome P-450 system inducers on the development of Drosophila melanogaster.

D. melanogaster development was markedly retarded and its survival decreased by larvae treatment with compounds being strong inducers of the cytochrome P-450 2B in mammals--phenobarbital (PB*), perfluorodecaline (PFD), transtilbene oxide (TSO), and triphenyldioxane (TPD). At the same time, the weak inducer hexobarbital or the selective cytochrome P-450 inducer in mice but not in rats 1,4-bis[2-(dichloropyridyl-oxy)]-benzene (DPB) did not affect the larvae development. The cytochrome P-450 1A1 inducers benzo(a)anthracene (BA) and beta-naphtoflavone (BNF) were also not effective. The toxicity of phenobarbital was shown to be decreased by the cytochrome P-450 inhibitor piperonyl butoxide by adding 20-hydroxyecdysone or by treatment with aminophylline--the indirect enhancer of ecdysone production in the larval prothoracic gland. The hypothesis of the moulting hormone degradation as the cause of elevated larvae mortality resulting from the induced high mixed function oxidase activity has been discussed.

The evaluation of methapyrilene for bacterial mutation with metabolic activation by Aroclor-induced, methapyrilene-induced and noninduced rat-liver S9

The antihistamine methapyrilene (MP) has been shown to be a potent hepatocarcinogen in rats. However, it has demonstrated little genotoxic activity in a wide variety of short-term tests. In this study, Fischer 344 rats were fed a carcinogenic dose of 0.1% methapyrilene in the diet for 10 weeks prior to sacrifice. S9 was prepared from the livers of the control, MP-treated and Aroclor-induced Fischer 344 rats. Each type of S9 was analyzed for mixed function oxidase activity, cytochrome P-450, and protein content. MP was then evaluated for mutagenicity in 6 strains of S. typhimurium (TA1535, TA1537, TA98, TA100, TA2638 and TA104) and one strain of E. coli (WP2uvrA −) using the standard plate-incorporation assay. MP was not mutagenic in any of the 7 bacterial strains when tested at concentrations ⩽ 10 mg/ml in the presence of each type of S9. However, in the absence of metabolic activation, an approximate 2-fold increase in revertants was noted with strain TA1535. The data from this study show that MP was not converted to a mutagenic metabolite by any of the three S9 types examined. However, the “weak” positive response with strain 1535 in the absence of metabolic activation indicates that further research is needed to elucidate the mechanism of action of this rat carcinogen.