Mixed Function Oxidase Activity Research Articles

Comparative Study on in vitro Inhibitory Effects of Heavy Metals on Rabbit Drug-Metabolizing Enzymes.

The in vitro inhibitory effects of heavy metals, i.e., mercury, cadmium, lead, nickel and beryllium in their chloride forms, on rabbit pulmonary drug-metabolizing enzymes were studied comparatively. Microsomal and cytosolic fractions prepared from rabbit lungs were incubated in the presence of heavy metals prior to enzyme assays. Mercury was the most potent in reducing cytochrome P450 content and mixed-function oxidase activities including NADPH-cytochrome c reductase and benzo[a]pyrene hydroxylase. The addition of mercury to pulmonary microsomal preparations resulted in a spectral shift from 450 nm to 420 nm in an absorption maximum of cytochrome P450carbon monoxide complex with mercury chloride being more potent than methylmercury. Cadmium was also inhibitory, while the effects of nickel were noted only at higher concentrations. Neither lead nor beryllium was inhibitory. Among second-phase drug-metabolizing enzymes, UDP-glucuronyltransferase and glutathione S-transferase were found to be susceptible to the adverse effects of mercury and lead, respectively. The extent of inhibition of the latter activity by mercury were highly dependent on the concentration of glutathione, implying the complex formation between them. Cadmium was slightly inhibitory to both enzyme activities, though the other metals had no effect. The results indicate that the pulmonary toxicities of airborne heavy metals could be inferred using simple in vitro assays.

Skin biopsy of Mediterranean cetaceans for the investigation of interspecies susceptibility to xenobiotic contaminants

Various studies on Mediterranean cetaceans have revealed bioaccumulation of contaminants such as organochlorines (OCs) and heavy metals. The susceptibility of these animals to organic pollutants and the relationship between bioaccumulation and population decline (as in the case of Delphinus delphis) are unexplored fields. In this study, we used a non-destructive approach (skin biopsy) to explore OC bioaccumulation processes and mixed-function oxidase activity (BPMO) in four species of cetaceans: striped dolphin ( Stenella coeruleoalba), bottlenose dolphin ( Tursiops truncatus), common dolphin ( D. delphis) and fin whale ( Balaenoptera physalus). Significant differences in BPMO induction and OC levels were found between odontocetes and mysticetes, the former having mixed-function oxidase activities four times higher than the latter, binding with levels of OCs one order of magnitude higher in odontocetes. A significant correlation ( P<0.05) between BPMO activities and OC levels was found in B. physalus. In an ongoing project, fibroblast cultures have been used as an alternative in vitro method of evaluating interspecies susceptibility to contaminants such as OCs and polycyclic aromatic hydrocarbons (PAHs). These results suggest that cetacean skin biopsies are a powerful non-invasive tool for assessing ecotoxicological risk to Mediterranean marine mammals species.

Specific toxins destabilize virus inhibitors (e.g. AIDS viruses)

There are two approaches to the study of viral infections – the A–Z hypothesis of Sprietsma involving thiol-zinc inhibition of protease activation in the virus coat, and the activity of toxins in depleting the cell of thiols, or the activity of toxins activating (not detoxifying) the mixed function oxidase (MFO) system. Both of these systems generate free radicals and utilize thiols in the process.Zinc forms stable mercaptides with thiols, inhibits cyclic reduction–autoxidation of thiols and superoxide generation. When the MFO system acts as an activator and not as a detoxifier in that intermediate products are more toxic than the original compound, zinc inhibits the oxidation. An example of increased toxicity with increased MFO activity is the toxin 3-methylindole (3-MI), a toxic product of intestinal bacterial putrifaction which reactivates the infectious bovine rhinotracheitis virus (IBR).Zinc reduces MFO activity and in this regard it functions synergistically with antioxidants in protecting cell membranes.It is hypothesized that stable zinc complexes inhibit activity of proteases in the virus nucleocapside (NC) proteins in the virus coat, both directly and indirectly because zinc also inactivates some toxins that are thiol depleters or virus reactivators.

A Study of the Acid Lability and Acute Toxicity of Dimethoxymethane in Rats

The pH stability and acute oral toxicity of dimethoxymethane (DMM) in rats were studied. In in vitro studies, DMM was found to be stable in artificial gastric juice at pH 2.5 and above but readily hydrolyzed at pH 2 and below, with 1 mole of DMM producing approximately 1 mole of formaldehyde and 2 moles of methanol. DMM incubated in sealed, excised stomachs appeared to equilibrate with the external incubation fluid over a 24-hour period without significant hydrolysis. In the toxicity study, three groups of female rats ( n = 5) were administered, by gavage, phosphate-buffered saline (control), DMM (31 mmoles/kg body weight [bw]/day), or methanol (MeOH) (30 mmoles/kg bw/day), respectively, for 3 consecutive days and then sacrificed on day 5. Transient ataxia lasting about 30 minutes was observed in animals following DMM administration. Increased urine volume was observed in the DMM group at the end of days 4 and 5. Markedly elevated level of formic acid was present in the urine of the MeOH group at days 1, 2, and 3, but not in the DMM group. Urinary ascorbic acid levels were increased in the DMM group on days 1, 2 and 3, and returned to baseline level at days 4 and 5. Microscopic examination detected no abnormal histological changes in the liver and kidneys of MeOH-or DMM-treated animals. No treatment effects were observed on the following endpoints: organ weights (liver, heart, thymus, kidneys), serum corticosterone, serum chemistry profile, hematology, hepatic phase 1 and phase 2 mixed-function oxidase activities, hepatic and serum thiobarbituric acid, hepatic glutathione, urinary protein, and urinary N-acetylglucosaminidase activity. These observations suggested that hydrolysis of DMM in the stomach required a gastric acidity below pH 2.5. At higher pH, intact DMM readily penetrated the stomach and therefore likely reached the circulation and other body tissues. There was no significant hydrolysis of orally administered DMM, and the acute effects were limited to transient ataxia and biochemical responses in the liver.

Antioxidant status, lipid peroxidation, mixed function oxidase and UDP-glucuronyl transferase activities in livers from control and DOCA-salt hypertensive male Sprague Dawley rats.

The effects of DOCA-salt hypertensive treatment on hepatic glutathione-dependent defense system, antioxidant enzymes, lipid peroxidation, mixed function oxidase and UDP-glucuronyl transferase activities were investigated in male Sprague Dawley rats. Compared with controls, DOCA-salt hypertensive rats had lower body weights (linked to liver hypertrophy). Mixed function oxidase and p-nitrophenol-UGT activities were not affected by the treatment but a significant lower rate of the glucuronoconjugation rate of bilirubin (p < 0.001) was observed in DOCA-salt hypertensive rats. While cytosolic glutathione contents and glutathione reductase activity were not affected, glutathione peroxidase (p < 0.001), glutathione transferase (p < 0.001) and catalase (p < 0.01) activities were decreased and associated with higher malondialdehyde contents (p < 0.001) in treated rats. The imbalance in liver antioxidant status (increasing generation of cellular radical species), associated with increases in lipid peroxidation, suggests that oxidative stress might be directly related to arterial hypertension in DOCA-salt treated male Sprague Dawley rats.

The effect of repeat administration of GTS-21 on mixed-function oxidase activities in rat

The effect of repeat administration of GTS-21 on hepatic microsomal enzymes was determined in rats administered the drug at levels of 3, 60 and 300 mg/kg/day for 7 days. Liver weight and cytochrome P450 (CYP) contents were not changed. Cytochrome b 5 contents were increased at the mid and high doses of GTS-21, as the contents increased with increasing dose, but were unchanged at the low dose. Five selective activities of CYP isoforms, acetanilide hydroxylase (CYP1A2), tolbutamide hydroxylase (CYP2C6), dextromethorphan O-demethylase (CYP2D1), p-nitrophenol hydroxylase (CYP2E1) and erythromycin N-demethylase (CYP3A) were examined. Enzyme activities were changed only at the highest dose; the activity of CYP1A2 was increased by 71% and the activities of CYP2C6 and CYP2D1 were decreased by 37 and 19%, respectively. At low and mid doses of GTS-21, all activities were unchanged. These data indicate that GTS-21 is not a strong modulator of the mixed-function oxidase system.

Induction of xenobiotic metabolising enzymes in the common brushtail possum, Trichosurus vulpecula, by Eucalyptus terpenes

The mixed function oxidase (MFO) activity and content was studied in the liver of common brushtail possums fed for 10 days on a diet containing a mixture of terpenes found in Eucalyptus leaves on which possums commonly browse. The MFOs were compared to the MFOs in possums fed a control diet of fruits and cereals only. The terpenes chosen were 1,8-cineole, p-cymene, α-pinene and limonene. The selected terpenes caused induction of P450 enzymes, as shown by a 53% higher cytochrome P450 content and a 45% increase in aminopyrine demethylase activity in the test animals. Aniline hydroxylase activity was significantly increased, with levels of 2.95 and 1.43 nmol min −1 mg −1 microsomal protein in the test and control animals, respectively. There was also a significant increase in androstenedione 16α-hydroxylase activity in the test group, 0.85 as compared to 0.50 nmol mg −1 min −1 in the control group. Western blot studies using human CYP2E1 and rat CYP2C11 and CYP2C6 antibodies gave CYP2E, CYP2C11 and CYP2C6 immunoreactive bands of greater intensity in the test animals as compared to the control group. This study has shown experimentally that dietary terpenes cause enzyme induction in folivorous marsupials. It also confirms the importance of knowledge of diet when studying xenobiotic metabolising enzymes, particularly in wild animals such as the brushtail possum.

EFFECT OF LOW-LEVEL SHORT-TERM o-XYLENE INHALATION ON BENZO[a]PYRENE (BaP) METABOLISM AND BaP-DNA ADDUCT FORMATION IN RAT LIVER AND LUNG MICROSOMES

o -Xylene is a commonly used solvent that alters mixed-function oxidase (MFO) activity in an organ- and isozyme-specific pattern following intraperitoneal (ip) administration. Similar MFO alterations have been observed after ip or inhalation exposure to other methyl benzenes. These MFO alterations shifted the metabolism of the carcinogen benzo[a]pyrene (BaP) toward formation of toxication metabolites in lung. The purpose of this study was to determine whether o-xylene inhalation caused similar MFO changes and whether these alterations were reflected in altered BaP metabolism and BaP-DNA adduct formation. o-Xylene (300 ppm, 6h) decreased the activity of arylhydrocarbon hydroxylase (AHH) in lung. CYP2B1 activity (benzyloxyresorufin O -dealkylase; BROD), which is responsible for metabolism of BaP to relatively nontoxic metabolites, was decreased in lung, as was, to a lesser extent, CYP1A1 (ethoxyresorufin O-dealkylase; EROD), which is responsible for metabolism of BaP to reactive/toxic metabolites. The BROD/EROD ratio, an indirect indicator of the pattern of BaP toxication/detoxication, was decreased in lung, suggesting that BaP metabolism is shifted toward toxication. No MFO alterations were observed in liver. In lung microsomes, o-xylene increased formation of 7,8-BaP-diol, while 9,10-BaP-diol, 3-OH BaP, and 9-OH BaP were decreased. In liver, o-xylene increased 9-OH BaP formation, while 4,5- and 9,10-diols as well as total diols were decreased. The toxication/detoxication ratios for BaP individual and total metabolite groups were increased in lung microsomes and unaltered in liver. The major BaP-DNA adduct, BaP diol epoxide-N 2-deoxyguanosine, was increased in lung but decreased in liver microsomes from o-xylene-exposed rats. Four minor BaP-DNA adducts were formed in lung and three in liver, only one of which (liver adduct 3) was decreased. The o-xylene-induced increase in BaP adduct formation in lung and decrease in liver indicate that coexposure to organic solvents such as the methyl benzenes may alter the carcinogenesis of BaP, or other PAHs, in an organ-specific fashion.

Phospholipase-induced modulation of rat liver mixed-function oxidase activity.

Conference Article| August 01 1999 Phospholipase-induced modulation of rat liver mixed-function oxidase activity C. E. Rolph; C. E. Rolph 1 1Department of Applied Biology, University of Central Lancashire, Preston, Lancs. PR1 2HE, U.K. 1To whom correspondence should be addressed Search for other works by this author on: This Site PubMed Google Scholar P. Roberts; P. Roberts 1Department of Applied Biology, University of Central Lancashire, Preston, Lancs. PR1 2HE, U.K. Search for other works by this author on: This Site PubMed Google Scholar S. Taylor S. Taylor 1Department of Applied Biology, University of Central Lancashire, Preston, Lancs. PR1 2HE, U.K. Search for other works by this author on: This Site PubMed Google Scholar Biochem Soc Trans (1999) 27 (4): 371–374. https://doi.org/10.1042/bst0270371 Article history Received: March 05 1999 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Cite Icon Cite Get Permissions Citation C. E. Rolph, P. Roberts, S. Taylor; Phospholipase-induced modulation of rat liver mixed-function oxidase activity. Biochem Soc Trans 1 August 1999; 27 (4): 371–374. doi: https://doi.org/10.1042/bst0270371 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search Search Dropdown Menu toolbar search search input Search input auto suggest filter your search All ContentAll JournalsBiochemical Society Transactions Search Advanced Search Keywords: NEFA, non-esterified fatty acid, PLA2, phospholipase A2 This content is only available as a PDF. © 1999 Biochemical Society1999 Article PDF first page preview Close Modal You do not currently have access to this content.

Expression of Cytochromes P-450 2E1, 3A4 and 1A1/1A2 in Growing and Confluent Human HepG2 Hepatoma Cells—Effect of Ethanol

In cultured human hepatoma HepG2 cells, cytochrome (CYP) 1A-associated 7-ethoxyresorufin- O-deethylase (EROD), CYP 3A-associated benzyloxyresorufin O-debenzylase (BROD) and CYP 2E1-associated p-nitrophenol-hydroxylase (PNPH) decreased during time in culture. The enzyme activities in cells at confluence were 35–60% of the activities in cells 24 hours after seeding. Similarly, CYP 3A and CYP 2E1 proteins were present at higher concentrations in growing (G) than in confluent (C) HepG2 cells. CYP 1A1/1A2 protein was not detected, neither in G nor in C HepG2 cells but was strongly induced by 3-methylcholanthrene (3-MC) treatment. Ethanol (EtOH) was shown to increase CYP 2E1 and CYP 3A proteins and CYP 1A1/1A2-, CYP 2E1- and CYP 3A-associated mixed-function oxidase activities (MFOs) in HepG2 cells, as has been previously reported for primary cultures of human hepatocytes. These effects were observed only at the beginning of culture, in growing HepG2 cells, demonstrating the influence of the growth stage of HepG2 cells on their response to EtOH treatment. This is, to our knowledge, the first report on increases in CYP proteins and associated MFOs by EtOH in HepG2 cells. It suggests that growing HepG2 cells provide a useful in vitro model system in which to study the regulation of human CYPs by EtOH.

The aminopyrine breath test (ABT) in a rat model of incomplete stenosis of the common bile duct

The hepatic cytochrome P-450 microsomal mixed function oxidase system that plays a major role in the metabolism of many endogenous and foreign compounds is one of the several liver functions impaired in total bile duct obstruction. However, no information existed concerning to the function of this system in incomplete bile duct stenosis, as seen in man in chronic pancreatitis, early stages of tumorous lesions of the common bile duct or in primary sclerosing cholangitis, in which the clinical features could be poor for a long time. In the present study, the microsomal mixed-function oxidase activity was tested in rats with incomplete mild stenosis of the common bile duct, by following weekly the aminopyrine breath test up to three weeks of evolution. The bile duct stenosis was evident from the increase of the biliary pressure, upstream dilatation of the bile duct, and ductular proliferation. Thus leaving the conduct intact, this model simulates a compression or constriction of the bile duct at early stages. The results showed a decrease in the peak of the exhalation and a lengthening of the half time of elimination of 14CO 2 from the metabolized 14C-aminopyrine that was injected, which were restored by the third week. This pattern was rather similar to that of serum bilirubin, which after an initial increase then returned to basal values. The study suggested that aminopyrine breath test, considered a measurement of the demethylase activity of the microsomal mixed function oxidase system, may be impaired in the incomplete stenosis of the bile duct. Likewise hepatic adaptive changes could support its recuperation.

Over-expression of cytochrome P450 CYP6B7 mRNA and pyrethroid resistance in Australian populations ofHelicoverpa armigera(Hübner)

Three cDNA clones for cytochrome P450s, CYP6B2, CYP6B6 and CYP6B7 which have 84-87% protein sequence identity have been isolated previously from Helicoverpa armigera, and the CYP6B7 mRNA was found to be over-expressed in a pyrethroid-resistant field population. Subsequent analysis has shown that over-expression is observed in a majority of individuals in all populations tested. Single-pair crosses between resistant and sensitive individuals indicated that the pyrethroid resistance co-segregated with the over-expression of this mRNA. Southern analysis indicated that the over-expression, which was confined to midgut only in many insects, was not related to gene amplification. These observations add weight to the conclusion that CYP6B7 is the cytochrome P450 form involved in pyrethroid resistance, and that over-expression of cytochrome P450 CYP6B7 is a common cause of pyrethroid resistance in H. armigera. The results suggest that specific probes for CYP6B7 protein or mRNA could provide the basis for the development of tools for monitoring pyrethroid resistance due to mixed function oxidase activity in field populations of this insect.

An evaluation of the relationships among oxidative stress, antioxidant vitamins and early mortality syndrome (EMS) of lake trout ( Salvelinus namaycush) from Lake Ontario

Mortality of the offspring in up to 50% of female lake trout from Lake Ontario appears just before swimup, and is known as early mortality syndrome (EMS). Adult lake trout from Lake Ontario also have high tissue concentrations of organic contaminants and elevated activities of hepatic MFO (mixed-function oxidase) biotransformation enzymes. Previous examinations of lake trout exposed to organochlorines in the laboratory and with elevated MFO activity have also identified increased indices of oxidative stress. We assayed MFO activity, as ethoxyresorufin- O-deethylase (EROD), and Ah-induced phase II activity, as glucuronyltransferase (UGT), in livers of adult female lake trout from Lake Ontario and from a reference population in Lake Manitou in order to examine relationships to oxidative stress. Enzymatic (superoxide dismutase, catalase, glutathione peroxidase) and non-enzymatic antioxidants (vitamins A, C and E), and oxidative membrane breakdown products (lipid hydroperoxides) were also quantified in livers of adult females. Eggs from each of the females were reared in the laboratory and analyzed at six time periods before the swimup stage for content of the antioxidant vitamins C and E and for vitamin A and its provitamin forms. MFO activity of the fry was assessed only at the postswimup stage. Adults whose offspring had EMS (>10% mortality) had lower content of vitamin E compared to the group from Lake Ontario without EMS (<10% mortality). Hepatic lipid hydroperoxides were elevated in both groups of adult lake trout from Lake Ontario. Except for total carotenoid concentrations, which were lower in eggs of lake trout from the EMS group, concentrations of antioxidant vitamins were comparable in eggs from lake trout with and without EMS from Lake Ontario and reference lake trout. No correlation was found among MFO activity, vitamin concentrations and the prevalence of EMS in lake trout fry at any of the developmental stages that were examined.

Relationship between organochlorine contaminants and mixed function oxidase activity in skin biopsy specimens of mediterranean fin whales ( Balaenoptera physalus)

The relationship between organochlorine contaminants (PCBs and DDTs) and mixed function oxidase, benzo(a)pyrene monooxigenase activity (BPMO), was investigated in skin biopsy specimens from fin whales ( Balaenoptera physalus) of the Mediterranean Sea. Skin biopsy material, sampled by a non invasive technique, is suitable for a wide range of chemical and biomarker analysis. In this study PCBs and DDTs were evaluated in subcutaneous blubber and MFO activity in epidermis. An interesting correlation was found in male specimens between the two variables.

Cytotoxicity of bile in human Hep G2 cells and in primary cultures of rat hepatocytes.

There has been increasing interest in the development of a hepatocyte bioreactor for the treatment of acute hepatic failure; however, little is known about the effect of hepatocyte byproducts on the viability of the cells in the bioreactor environment. We investigated the effects of increasing concentrations of bile on the growth and viability of the human hepatoma cell line Hep G2 and on the cytochrome P-450 content and dependent mixed function oxidase (MFO) activities, reduced glutathione (GSH) content, and glutathione S-transferase (GST) activity of primary cultures of rat hepatocytes. Our purpose was to determine whether or not it would be necessary to pretreat the plasma from patients with acute liver failure to remove elevated bile concentrations which might be toxic to the hepatocytes in an artificial liver device. Bile was found to inhibit Hep G2 cell growth at concentrations as low as 0.1% and to decrease viability at concentrations above 0.5%. The cytochrome P-450 and GSH contents and the activities of the MFO system and of GST were decreased in the primary cultures of hepatocytes following 24 h treatment with concentrations of bile at and above 0.5%. The MFO activities associated with different cytochrome P-450 isoenzymes decreased to different extents in the presence of bile with the O-dealkylation of pentoxyresorufin being more labile than that of ethoxyresorufin. Our data indicate that elevated bile concentrations are cytotoxic to liver cells, and it may be necessary to pretreat patient plasma to decrease its bile content to protect the cells during the clinical operation of a hepatocyte bioreactor device.

INHIBITION OF RAT LUNG MIXED-FUNCTION OXIDASE ACTIVITY FOLLOWING REPEATED LOW-LEVEL TOLUENE INHALATION: POSSIBLE ROLE OF TOLUENE METABOLITES

Toluene is a commonly used solvent that has been shown to alter mixed-function oxidase (MFO) activity, in an organ- and isozyme-specific pattern, following intraperitoneal administration. The purpose of this study was to determine whether similar changes occurred following repeated, low-level inhalation exposure, and to investigate the role of toluene metabolites in these alterations. Exposure to 375 ppm toluene, 6 h/d for up to 5 d, resulted in significant inhibition of the activity of pulmonary arylhydrocarbon hydroxylase (AHH), cytochrome P-4502B1 (CYP2B1), and CYP4B1, but not CYP1A1. After exposure to lower toluene levels (125 ppm, 6 h/ d, 3 d), the activities of lung AHH, CYP2B1, and CYP4B1 were also significantly decreased, but in a dose-related manner. MFO activity was not consistently altered in liver. Control pulmonary or liver microsomes were incubated with various concentrations (0.01-10 m M) of toluene or its metabolites and CYP2B1, CYP1A1, and/or CYP4B1 activities were subsequently determined. Benzaldehyde produced a significant dose-related inhibition in the activity of all three lung P-450s examined (IC50 10-3 M). Toluene was found to be a more potent inhibitor of lung CYP2B1 and CYP1A1 (IC50, 10-4 M) than benzaldehyde, but neither toluene nor benzyl alcohol was an effective inhibitor of lung CYP4B1. Toluene and its metabolites were weaker inhibitors of CYP1A1 than of CYP2B1. For CYP2B1 and CYP1A1, the order of inhibitory potency was toluene > benzaldehyde > benzyl alcohol and suggests that both the parent molecule and its metabolites may act in concert to inhibit catalytic activity of these cytochromes. The MFO inhibition seen after repeated low-level toluene inhalation exposure could result in altered metabolic profiles of other xenobiotics in an organ-specific fashion.

Assessment of Liver Function in Primary Cultures of Hepatocytes Using Diethoxy (5,6) Chloromethylfluorescein and Confocal Laser Scanning Microscopy

A method is presented which can be used to assess the function of hepatocytes in complex culture configurations without disrupting the integrity of the cell environment. It utilises a fluorescent probe for cytochrome P450 dependent mixed function oxidase (MFO) activity diethoxy (5,6) chloromethylfluorescein, and confocal laser scanning microscopy. The MFO activity of individual cells in primary cultures of intact hepatocytes can be detected in situ, and quantified by image analysis. This may be a valuable means of monitoring the effect of culture conditions on the function of bioartificial liver devices, and could be used to assess the need for effective oxygenation of cells, the influence of shear stress and of exposure to patient serum during clinical use.

Identification of chloro‐nitro‐trifluoromethyl‐substituted dibenzo‐p‐dioxins in lampricide formulations of 3‐trifluoromethyl‐4‐nitrophenol: Assessment to induce mixed function oxidase activity

AbstractThe contamination of field formulations of the lampricide 3‐trifluoromethyl‐4‐nitrophenol (TFM) bya dibenzo‐p‐dioxin containing ‐chloro, ‐nitro, and ‐trifluoromethyl substituents was suspected from chemical fractionations of a TFM formulation that were directed by mixed function oxidase (MFO) induction in rainbow trout. Three dioxin isomers containing these substituents in field formulations were identified by gas chromatography–mass spectrometry (GC‐MS). Short‐term waterborne exposures to a mixture of two isomers, 2‐trifluoromethyl‐3‐nitro‐7‐ (and 8)‐chloro‐dibenzo‐p‐dioxin, elevated MFO activity in trout, with a threshold between 0.148 and 0.745 nM (4.1–20.5 ng/L). Synthetic preparations of other dioxins related to formulation impurities enabled characterizations of this previously unknown family of dioxin congeners by GC‐MS. The elution order of the isomers followed those established for halogenated dioxins except where there was a lone ‐nitro substitution on one ring. The average concentration of these compounds in TFM formulations spanning more than a decade was 288 ± 47 μg/L, which translates into an annual loading of approximately 40 g to the Great Lakes Basin. Using mammalian (H4IIE) and fish (PLHC‐1) in vitro assays, a 2,3,7‐substituted chloro‐nitro‐trifluoromethyl isomer was a four to five times more potent inducer of MFO activity than 2,3,7‐trichlorodibenzo‐p‐dioxin. Characterizations of the synthetic isomers indicate that the isomers present in the formulation are not substituted in the 2,3,7‐ positions and are relatively weak inducers of MFO activity.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) Induces Hepatic Cytochrome P450-Dependent Arachidonic Acid Epoxygenation in Diverse Avian Orders: Regioisomer Selectivity and Immunochemical Comparison of the TCDD-Induced P450s to CYP1A4 and 1A5

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) markedly induced cytochrome P450 (CYP)-dependent arachidonic acid metabolism in liver microsomes from hatchlings of four avian species belonging to four different orders: chick, pigeon, cormorant, and great blue heron, increasing formation of arachidonic acid epoxides (EETs), monohydroxyeicosatetraenoic acids (HETEs), ω−1, and ω−2 OH arachidonic acid products by fivefold or more. Microsomes from TCDD-induced hatchling chicks had the highest activity and the least restricted EET regioselectivity. ω-OH arachidonic acid, the principal constitutive metabolite in chick and pigeon liver microsomes and a major product for cormorant and great blue heron was not induced by TCDD. Constitutive EET formation in avian liver microsomes was very low except in cormorant microsomes where 8,9-EET was generated almost exclusively. Western blots of liver microsomes using polyclonal antisera to chick embryo-derived CYP1A4 and 1A5 recognized two TCDD-induced bands in each of the species. The chick bands had the same molecular weights as CYP1A4 and 1A5 (55 and 55.5 kDa, respectively) but those of the other species differed. Immunopurified antiserum monospecific for CYP1A5 recognized a band in microsomes from all of the avian species, and monospecific antiserum for CYP1A4 recognized a band in microsomes from chick, pigeon, and great blue heron. AntiCYP1A4 and 1A5 IgG immunoinhibited TCDD-induced mixed function oxidase activity completely in chick and chick embryo microsomes and only partially in the other avian microsomes. The results demonstrate that (1) TCDD causes much greater induction of CYP-dependent arachidonic acid metabolism, and of arachidonic acid epoxygenation in particular, in avian than in mammalian species; (2) TCDD induces two CYP1A-related enzymes in birds as in mammals; (3) CYP1A enzymes in the birds other than chicks are not identical to CYP1A4 and 1A5 but share some enzymatic and immunochemical characteristics with them; (4) constitutive ω-OH arachidonic acid in all of the avian species and 8,9-EET in cormorant are formed by CYP enzymes unrelated to CYP1A; and (5) two distinct characteristics of avian CYP1A enzymes are the acquisition by avian CYP1A4-related P450 of unique epitope(s) and by CYP1A5-related P450 of unusual catalytic effectiveness for arachidonic acid epoxygenation.

The Activity of Mixed Function Oxidases, Estimated by in vivo Antipyrine Clearance, Is Similar in Horses and Camels

The activity of hepatic mixed function oxidases was compared in horses and camels ( Camelus dromedarius) by studying the pharmacokinetics of antipyrine in seven camels and five horses following intravenous administration of a single dose of antipyrine (25 mg/kg). The data obtained (mean ± SEM and median in brackets) in camels and horses, respectively, were as follows: the elimination half-lives were 3.25 ± 0.23 (3.19) and 3.09 ± 0.25 (2.90) hr; the apparent volumes of distribution (area method) were 0.691 ± 0.0 45 (0.648) and 0.642 ± 0.0 34 (0.676) l/kg; the volumes of distribution at steady state were 0.659 ± 0.0 40 (0.607) and 0.620 ± 0.0 30 (0.653) l/kg; the volume of the central compartment of the two-compartment pharmacokinetic model were 0.386 ± 0.0523 (0.349) and 0.298 ± 0.05 (0.308) l/kg; total body clearances were 0.148 ± 0.008 (0.158) and 0.145 ± 0.007 (0.147) l/kg/hr; the areas under the curves to infinity were 171.0 ± 9 (165) and 175 ± 8.0 (170) μg. ml. hr. There was no statistical significance in any parameter between camels and horses which suggests that the activity of hepatic mixed function oxidases is similar in horses and camels.