Mixed Function Oxidase Enzyme Research Articles

Short exposure to nitenpyram pesticide induces effects on reproduction, development and metabolic gene expression profiles in Drosophila melanogaster (Diptera: Drosophilidae)

Although the toxicity of neonicotinoid insecticides has been demonstrated in several studies, the information on metabolism, behavior, and health risk remains limited and has raised concerns about its potential toxicity. Thus, in this study we assessed the effects of nitenpyram using different sublethal concentrations (one-third and one-tenth of the acute LC50 values) on various developmental and metabolic parameters from gene expression regulation in Drosophila melanogaster (model system used worldwide in ecotoxicological studies). As a result, nitenpyram sublethal concentrations prolonged the developmental time for both pupation and eclosion. Additionally, nitenpyram sublethal concentrations significantly decreased the lifespan, pupation rate, eclosion rate, and production of eggs of D. melanogaster. Moreover, the mRNA expression of genes relevant for development and metabolism was significantly elevated after exposure. Mixed function oxidase enzymes (Cyp12d1), (Cyp9f2), and (Cyp4ae1), hemocyte proliferation (RyR), and immune response (IM4) genes were upregulated, whereas lifespan (Atg7), male mating behavior (Ple), female fertility (Ddc), and lipid metabolism (Sxe2) genes were downregulated. These findings support a solid basis for further research to determine the hazardous effects of nitenpyram on health and the environment.

Toxicity of Essential Oils Nanoemulsion Against Aphis Craccivora and Their Inhibitory Activity on Insect Enzymes

Essential oils are widely used as botanical insecticides rather than chemically synthesized pesticides which led to catastrophic effects on humans, the environment, and eutrophication. Here, encapsulation of four essential oils Basilicum ocimum, Cuminum cyminum, Origanum marjorana, and Matricaria chamomilla were utilized in the presence of 3% v/v ethanol, as anti-insect against Aphis craccivora and compared to traditional insecticides dinotefuran and pymetrozine. Different tools were used to characterize the prepared nanoemulsion such as TEM, SEM, and Zeta potential analyzer. Besides, selected B. ocimum and C. cyminum were analyzed by gas chromatography-mass GC/mass spectrometry. The results reveal that nanoemulsion exhibited considerable toxic activities against laboratory and field strains of cowpea aphid. In the toxicity bioassay test of essential oils, moderate mortality was observed at 10,000 mg/L against aphid with lethal concentration that kills 50% of insects (LC50) values of basil 992 mg/L and marjoram 3162 mg/L. Else, nanoemulsion provided the highest mortality rate at 625 mg/L and the LC50 values of basil nanoemulsion (NE) 45 mg/L, and marjoram NE 188 mg/L in laboratory strains. The systemic effects of the tested substances acetylcholine esterase, alkaline phosphatase, β-esterases, glutathione S-transferase (GST), and mixed-function oxidase (MFO) enzymes on insects were found to be significantly decreased and increased when compared with control groups. Overall, these results highlight that the nanoemulsion is potential tools to control cowpea aphid and could be useful in developing integrated insect management in faba bean fields.

Hazardous effects of octopamine receptor agonists on altering metabolism-related genes and behavior of Drosophila melanogaster

Recent reports demonstrate that octopamine receptor (OR) agonists such as formamidine pesticides cause reproductive and developmental toxicity through endocrine disrupting effects in both humans and animals. Herein, we studied the effects of different sublethal concentrations of OR agonists, Amitraz and Chlordimeform, on growth, development, and reproduction of D. melanogaster from a genotype perspective view. As a result, the sublethal concentrations for both OR agonists delayed the developmental time including pupation and eclosion. It significantly reduced the lifespan, eclosion rate, and production of eggs. The mRNA expression of genes relevant for development and metabolism was significantly changed after exposure to sublethal concentrations of both OR agonists. Octopamine receptor in mushroom bodies (Oamb), trehalase enzyme (Treh), hemocyte proliferation (RyR), and immune response (IM4) genes were upregulated whereas, trehalose sugar (Tret1-1), mixed function oxidase enzyme (Cyp9f2), lifespan (Atg7), male mating behavior (Ple), female fertility (Ddc), and lipid metabolism (Sxe2) genes were downregulated. These results support the conclusion that OR agonists activate the octopamine receptor in D. melanogaster leading to an increase of trehalase enzyme activity and degradation of trehalose sugar into free glucose which results in rapid energy exhaustion, hyperexcitation, and disturbing of the octopaminergic system in D. melanogaster.

Molecular mechanisms of the microsomal mixed function oxidases and biological and pathological implications

The cytochrome P450 mixed function oxidase enzymes play a major role in the metabolism of important endogenous substrates as well as in the biotransformation of xenobiotics. The liver P450 system is the most active in metabolism of exogenous substrates. This review briefly describes the liver P450 (CYP) mixed function oxidase system with respect to its enzymatic components and functions. Electron transfer by the NADPH-P450 oxidoreductase is required for reduction of the heme of P450, necessary for binding of molecular oxygen. Binding of substrates to P450 produce substrate binding spectra. The P450 catalytic cycle is complex and rate-limiting steps are not clear. Many types of chemical reactions can be catalyzed by P450 enzymes, making this family among the most diverse catalysts known. There are multiple forms of P450s arranged into families based on structural homology. The major drug metabolizing CYPs are discussed with respect to typical substrates, inducers and inhibitors and their polymorphic forms. The composition of CYPs in humans varies considerably among individuals because of sex and age differences, the influence of diet, liver disease, presence of potential inducers and/or inhibitors. Because of such factors and CYP polymorphisms, and overlapping drug specificity, there is a large variability in the content and composition of P450 enzymes among individuals. This can result in large variations in drug metabolism by humans and often can contribute to drug–drug interactions and adverse drug reactions. Because of many of the above factors, especially CYP polymorphisms, there has been much interest in personalized medicine especially with respect to which CYPs and which of their polymorphic forms are present in order to attempt to determine what drug therapy and what dosage would reflect the best therapeutic strategy in treating individual patients.

Methodology to assay CYP2E1 mixed function oxidase catalytic activity and its induction

The cytochrome P450 mixed function oxidase enzymes are the major catalysts involved in drug metabolism. There are many forms of P450. CYP2E1 metabolizes many toxicologically important compounds including ethanol and is active in generating reactive oxygen species. Since several of the contributions in the common theme series “Role of CYP2E1 and Oxidative/Nitrosative Stress in the Hepatotoxic Actions of Alcohol” discuss CYP2E1, this methodology review describes assays on how CYP2E1 catalytic activity and its induction by ethanol and other inducers can be measured using substrate probes such as the oxidation of para-nitrophenol to para-nitrocatechol and the oxidation of ethanol to acetaldehyde. Approaches to validate that a particular reaction e.g. oxidation of a drug or toxin is catalyzed by CYP2E1 or that induction of that reaction is due to induction of CYP2E1 are important and specific examples using inhibitors of CYP2E1, anti-CYP2E1 IgG or CYP2E1 knockout and knockin mice will be discussed.

Medicinal Chemistry of Drugs with Active Metabolites (N-, O-, and S-desalkylation and Some Specific Oxidative Alterations)

Metabolic fate plays an important role in the elimination of drugs and other foreign compounds from the body. Metabolism through various enzyme systems, makes the parent compound more hydrophilic, thus, it can be readily excreted from the body. Some active metabolites of drugs are produced following N-, O-, and S-desalkylation. These metabolites are either more or less potent, or as potent as their parent drugs. The removal of alkyl groups from tertiary aliphatic and acyclic amines is carried out by hepatic cytochrome P450 mixed-function oxidase enzymes. Several drugs undergo this process, which yields free hydroxyl-, or amino-groups, in addition to aldehyde from the splitted alkyl group. Metabolism of drugs into clinically active compounds indicates an extra target of therapeutic drug monitoring. Numerical data of logP values show how lipophilicity changes through metabolism to facilitate excretion. The example of phenacetin - paracetamol opened up a way for improving pharmacological effect by the use of a metabolite. This review gives a detailed description of these drugs, their active and major metabolites found in humans or animals, metabolizing cytochrome P450s, and the most recent analytical methods for their determination.

Susceptibility of field populations of Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) in China to chlorantraniliprole and the activities of detoxification enzymes

The cutworm Spodoptera litura is a major insect pest of vegetables and cotton in China, and has been reported to develop resistance to various classes of insecticides. Chlorantraniliprole, which has been registered recently in China, provides a novel option for control of this pest. The susceptibilities of S. litura collected from Southeast China to chlorantraniliprole were determined by diet incorporation assay with neonates. The susceptibility variation among 12 field populations was low (<4-fold), with median lethal concentration (LC50) values varying from 28.4 to 102.5 μg/l. However, all the 12 field populations were less susceptible to chlorantraniliprole than a laboratory susceptible population. The most tolerant populations were sampled from Guangdong and Anhui Provinces where S. litura had been frequently challenged by insecticides. However, no correlation was found between LC50 values and the number of applications of this chemical. Biochemical assays were performed to determine the potential mechanisms involved in the tolerance observed in field populations. Most field populations showed significantly enhanced activities of mixed function oxidase enzymes compared with the susceptible strain. Only a few populations had higher activities of esterase and glutathione-S-transferase than the susceptible strain. No correlations were observed between activities of metabolic enzymes and chlorantraniliprole toxicity, suggesting that these detoxification enzymes were not the main cause of the field tolerance observed in this study, and there might be other mechanisms conferring the tolerance variation to chlorantraniliprole in S. litura.

Biochemical effects of some agricultural waste extracts against Culex pipiens (Diptera: Culicidae)

Third larval instar of Culex pipiens were exposed to sub-lethal concentration LC25 of Black and White liquors (waste extract from rice straw) for 24 hours to study its effect on some enzymes activity along different developmental stages of mosquito.Acetylcholinesterase (AchE) enzyme showed high activity in all stages of mosquito treated in the larval stage with black and white liquors. Treatment with black and white liquors showed lower in enzyme activity of esterases (α-est, β-est) and Glutathione s-transferase (GST). While, mixed function oxidase (MFO) enzyme showed high activity in all developmental stages of C. pipiens larval treatment with black and white liquors in comparing with control except in pupal stage low activity and not affected, respectively.

Permethrin resistance in Aedes aegypti (Linnaeus) collected from Kuala Lumpur, Malaysia

Permethrin resistance status of a laboratory strain, a permethrin-selected strain and three field strains of Aedes aegypti collected in Kuala Lumpur, Malaysia were evaluated using three standard laboratory bioassays: WHO larval bioassay, WHO adult mosquito bioassay, and mixed function oxidase (MFO) enzyme microassay. The LC 50 values of field strains from the WHO larval bioassay did not differ significantly. The highest LC 50 value was from the Taman Melati field strain (0.39 mg/L). The resistance ratio for the permethrin-selected strain and the field strains ranged from 1.86 fold to 5.57 fold. Pre-exposure to piperonyl butoxide (PBO) in the WHO adult bioassay and MFOs enzyme microassay reduced the LT 50 values and reduced the mean optical density of elevated oxidase activity (0.28–0.42) at 630 nm. The LC 50 or LT 50 values and the level of oxidases were significantly correlated ( r = 0.825; p< 0.05). This study confirmed the presence of permethrin resistance in these mosquito populations.

Intravenous neonatal paracetamol dosing: the magic of 10 days

Intravenous (i.v.) paracetamol (acetaminophen) is gaining increasing use in neonates (1–3), despite an off-label status in some countries. Product information for i.v. paracetamol does not consider the clearance maturation with age in neonates in its dosing recommendations. In Australasia the same dose of 15 mgAEkg 6 h is suggested for neonates over 10 days of age and for infants and children up to 33 kg. A reduced dose of 7.5 mgAEkg 6 h is proposed in term neonates <10 days of age. In Europe there is no labeled use for neonates <10 days of age. The registered dose is 7.5 mgAEkg 6 h for neonates after 10 days of age through to infants of 10 kg. A dose of 15 mgAEkg 6 h (max daily dose 60 mgAEkg) is recommended between 10 and 40 kg. These dosing regimens suggest something magic about 10 days. There are data concerning the pharmacokinetics of this formulation in neonates. Population pharmacokinetics of the i.v. prodrug (propacetamol) have been studied in neonates following single (4,5) and repeat dose administration (6). Parameter estimates from neonates given repeat doses of i.v. paracetamol over a median of 4 days duration are consistent with those estimated using propacetamol (7). Dosing regimens used in some institutions for neonates have been published in Pediatric Anesthesia (2,8), but there are few data validating the safety or effectiveness of these regimens. The idea that a target effect and associated target concentration can be used to make rational individual dose decisions has acceptance (9). A knowledge of neonatal pharmacokinetics assists with dose prediction to achieve the target concentration. There are, however, flaws and constraints of current knowledge that limit implementation of this approach to dosing.

Phase I Enzyme Induction in Girardinichthys viviparus, an Endangered Goodeid Fish, Exposed to Water from Native Localities Enriched with Polychlorinated Biphenyls

The present study examines the induction of mixed-function oxidase (MFO) enzymes, including CYP content CYP1A (EROD) activity and alcohol dehydrogenase activity (ADH), in an endemic Mexican fish species, the black-fin goodeid Girardinichthys viviparus, exposed to the water of two localities, Lake Texcoco (LTX) and Lake Zumpango, and to the same matrices enriched in polychlorinated biphenyls (PCBs) to simulate the potential toxic effects of sublethal increases in these xenobiotics. Fishes of both sexes born in the laboratory were exposed for 1, 2, 4, 8, and 16 days. Water from the two types of localities of the black-fin goodeid contains MFO inducers. Of the two, the most contaminated is LTX water, which also contains PCBs. EROD activity was higher in all treatments with female compared with male fish. This suggests greater metabolic compromise in female fish as a response to damage caused by these xenobiotics. In this species, CYP induction displayed two patterns that were not always concurrent with higher CYP1A activity. In the enriched matrix system, biotransformation processes were notably altered. Increased ADH may indicate that this enzyme is involved in the biotransformation of PCBs and their metabolites, particularly in male fish, and provides at least a part of reductive power required by the MFO enzymes; however, specific studies are needed to clarify this point.

Modification of pesticide toxicants Actara, Desis and Fabcosidine for larvae of Khabra Trogoderma granarium beetle with Prenobyl Piotoxide and phenobarbital

This study was designed to evaluate the toxicities of Actara, Decis and Vapcocidin and the effects of the mixed-function oxidase (mfo) enzymes inhibitor, piperonyl butoxide (pb ), and inducer, Phenobarbital (phb ), on the toxicities to the 15 2nd, 3rd, 4th and 51h instar larvae of Trogoderma granarium, using the impregnated filter paper technique. Based on the 24h LC50 values, the three insecticides were highly toxic to the larvae. Decis was about twice as toxic as Actara and both were greatly much more toxic than Vapcocidin. The toxicities of the three insecticides, generally, decreased as the Larval stage advanced (LC50 values in the 5- Larval instars were 1.7, 1.8, 2.2, 2.6, 3.1 0 l/filter paper, respectively, for Decis, 3.0, 3.5, 4.5, 6.0, 7.0 0 1/filter paper, respectively, for Actara and 6.0, 8.0, 11.0, 13.0, 15.0 0 1/filter paper, respectively, for Vapcocidin) and it is considered most likely that the decrease is due to the increase in the mfo enzymes activity. The mfo enzymes modifiers data showed that pband pbb greatly increased and decreased, respectively, the toxicities of the three insecticides. The date also revealed that mfo insecticide detoxifing enzymes activity increased as the Larval stage advanced. The studysuggests that Actara would be an effective alternative to pyrethroids against this insect.

Elimination of major side effects due to ROS of therapeuticals through biotransformation control of the 57 cytochromes P450 isoenzymes

Drug biotransformations are controlled in the body by tissue-differentiated choice of a selection of the induced and constitutive 57 isoforms of the human cytochromes P450 (CYP). CYP are the mixed function oxidase enzyme family (EC 1.14.14.1: mono-oxygenases that contain iron-dependent protohaematin IX). Many of the major side effects of clinical therapeutical agents have been attributed to the generation of highly toxic "reactive oxygen species" (ROS). Such ROS generation can be circumvented however by the ability to exert control over which forms of CYP are present: and as to which are bioactive in tissues other than the drug-target ones. Up to 57 isoenzymes of human cytochromes P450 are likely to be present: generation of a variety of species of ROS can be anticipated therefore with outcomes that include cancer, premature ageing, heart (arterial) damage and osteoporosis (in association with oestrogen/testosterone imbalance). Moreover, therapeutic vaccines could be constructed to remove the most harmful forms of cytochromes P450. Designer CYP protein enzymes can now be readily constructed by genetic engineering techniques: these include site-directed mutagenesis to place the modified cyp gene in suitable enzyme expression location in particular tissues only. Furthermore, gene therapy could be arranged to augment (or replace activities lost) for example the production of insulin in the Islets of Langerhans of the pancreas by production of the appropriate form of human insulin in the liver.

Mixed Function Oxidase Activity and Organochlorine Levels in Farmed Sharpsnout Seabream (Diplodus puntazzo) from Two Intensive Aquaculture Facilities

Mixed function oxidase (MFO) activities and organochlorine levels were investigated in liver of farmed sharpsnout seabream bred in two intensive facilities, a sea farm and a land-based farm. The aim of this study is to investigate how different facility locations and breeding conditions might affect fish metabolic capabilities and accumulation of toxic chemicals in farmed sharpsnout seabream. The differences between breeding two or more fish species in the same cage or tank known as polyculture, and monoculture (1 species) were also investigated. The results showed that both facility location (sea and land) as well as breeding systems (polyculture and monoculture) might be responsible for the differences observed in both MFO enzyme activity and organochlorine levels. Significantly higher activities of two metabolizing enzymes such as 7-ethoxyresorufin-O-deethylase (EROD) and benzo(a)pyrene monooxygenase (B(a)PMO) were in fact recorded in land-based farmed fish than in sea farmed ones, and much higher in those from polyculture than in monoculture (p < 0.05). By contrast, the highest levels of dichlorodiphenyldichloroethilene (pp′DDE) and polychlorinated biphenyls (PCBs as Aroclor 1260) were measured in sea-farmed fish, except for hexachlorobenzene (HCB), which gave higher levels in land-based farmed fishes. No significant differences were observed between monoculture and polyculture in p,p′DDE and PCBs levels except for p,p′DDE which was significantly higher in monoculture than in polyculture (p < 0.05) in fish reared in tanks. Finally significantly higher HCB levels were measured in fish reared in monoculture in tanks (p < 0.05) and in polyculture in cages (p < 0.05). The low TEQ values obtained for PCB congeners at liver level indicated no concern for fish health.

Testing the ecotoxicology of vegetable versus mineral based lubricating oils: 2. Induction of mixed function oxidase enzymes in barramundi, Lates calcarifer, a tropical fish species

An increasing number of vegetable-based oils are being developed as environmentally friendly alternatives to petroleum products. However, toxicity towards key tropical marine species has not been investigated. In this study we used laboratory-based biomarker induction experiments to compare the relative stress of a vegetable-based lubricating oil for marine 2-stroke engines with its mineral oil-based counterpart on tropical fish. The sub-lethal stress of 2-stoke outboard lubricating oils towards the fish Lates calcarifer (barramundi) was examined using liver microsomal mixed function oxidase (MFO) induction assays. This study is the first investigation into the use of this key commercial species in tropical North Queensland, Australia in stress assessment of potential hydrocarbon pollution using ethoxyresorufin O-deethylase (EROD) induction. Our results indicated that barramundi provide a wide range of inducible rates of EROD activity in response to relevant organic stressors. The vegetable- and mineral-based lubricants induced significant EROD activity at 1.0 mg kg−1 and there was no significant difference between the two oil treatments at that concentration. At increasing concentrations of 2 and 3 mg kg−1, the mineral-based lubricant resulted in slightly higher EROD activity than the vegetable-based lubricant. The EROD activity of control and treated barramundi are found to be within ranges for other species from temperate and tropical environments. These results indicate that vegetable-based lubricants may be less stressful to barramundi than their mineral counterparts at concentrations of lubricant ⩾2 mg kg−1. There is great potential for this species to be used in the biomonitoring of waterways around tropical North Queensland and SE Asia.

Effect of the Degree of Hydrogenation of Fish Oil on the Enzymatic Activity and on the Fatty Acid Composition of Hepatic Microsomes from Young and Aged Rats

By modifying the degree of hydrogenation of dietary fat, it is possible to modify the fatty acid composition and the biochemical activity of cellular tissues. The age can be another variable influencing these modifications. The effect of isocaloric diets containing oils with different degrees of hydrogenation: fish oil (FO, 0.3% trans), partially hydrogenated fish oil (PHFO, 29% trans), or highly hydrogenated fish oil (HHFO, 2.3% trans), in the fatty acid composition (cis and trans isomers) of hepatic microsomes from young (70-day-old) and aged (18-month-old) rats, in the microsomal cytochrome P-450 (C-450) content, and in the aminopyrine N-demethylase (AND), aniline hydroxylase (AH), NADPH cytochrome P-450 reductase (NCR), UDP-glucuronyl transferase (UGT), and GSH-S transferase (GST) enzymatic activities were studied. Fatty acid composition and n–6/n–3 ratio of microsomal membranes was modified to a higher extent in young rats. C-450 content and AND activity were reduced when the degree of hydrogenation of dietary fat was increased in the young and the aged rats. AH activity was higher after the PHFO diet in the young rats only. NCR activity was reduced in the young animals when the hydrogenation of the fat was increased. However, in aged rats the enzyme exhibited a higher activity after the PHFO and HHFO diet. UGT and GST activities where not affected by the level of hydrogenation of the dietary fat in both the young and the aged rats. However, UGT activity was higher in the young rats, while GST activity was higher in the aged animals. We conclude that hydrogenation of dietary fat can modify the fatty acid composition of hepatic microsomes, young animals being more sensitive to these changes than aged animals. These effects were also reflected in the amount and/or the activity of some molecular components of the hepatic microsomal mixed-function oxidase enzyme system. Microsomal trans fatty acid composition is not affecting the activity of the enzymes, the age of the animals being the most important factor

Characterization of moclobemide N-oxidation in human liver microsomes

1. Moclobemide undergoes morpholine ring N-oxidation to form a major metabolite in plasma, Ro12-5637. 2. The kinetics of moclobemide N-oxidation in human liver microsomes (HLM) (n = 6) have been investigated and the mixed-function oxidase enzymes catalysing this reaction have been identified using inhibition, enzyme correlation, altered pH and heat pretreatment experiments. 3. N-oxidation followed single enzyme Michealis-Menten kinetics (0.02-4.0 mM). Km app and Vmax ranged from 0.48 to 1.35 mM (mean ± SD 0:77 ± 0:34 mM) and 0.22 to 2.15 nmol mg−1 min−1 (1:39 ± 0:80 nmol mg−1 min−1), respectively. 4. The N-oxidation of moclobemide strongly correlated with benzydamine N-oxidation, a probe reaction for flavin-containing monooxygenase (FMO) activity, (0.1 mM moclobemide, rS = 0:81; p < 0:005; 4 mM moclobemide, rS = 0:94; p = 0:0001). Correlationswere observed between moclobemide N-oxidation and specific cytochrome P450 (CYP) activities at both moclobemide concentrations (0.1 mM moclobemide, CYP2C19 rS = 0:66; p < 0:05; 4 mM moclobemide, CYP2E1 rS = 0:56; p < 0:05).5. The general P450 inhibitor, N-benzylimidazole, did not affect the rate of Ro12-5637 formation (0% inhibition versus control) at 1.3 mM moclobemide. Furthermore, the rate of Ro12-5637 formation in HLM was unaffected by inhibitors or substrates of specific P450s (<10% inhibition versus control). 6. Heat pretreatment of HLM in the absence of NADPH (inactivating FMOs) resulted in 97% inhibition of Ro12-5637 formation. N-oxidation activity was greatest when incubated at pH 8.5. These results are consistent with the reaction being FMO mediated. 7. In conclusion, moclobemide N-oxidation activity has been observed in HLM in vitro and the reaction is predominantly catalysed by FMOs with a potentially small contribution from cytochrome P450 isoforms.

Prodrugs of nitroxyl and nitrosobenzene as cascade latentiated inhibitors of aldehyde dehydrogenase.

The prototypic aromatic C-nitroso compound, nitrosobenzene (NB), was shown previously to mimic the effect of nitroxyl (HN=O), the putative active metabolite of cyanamide, in inhibiting aldehyde dehydrogenase (AlDH). To minimize the toxicity of NB in vivo, pro-prodrug forms of NB, which were designed to be bioactivated either by an esterase intrinsic to AlDH or the mixed function oxidase enzymes of liver microsomes, were prepared. Accordingly, the prodrug N-benzenesulfonyl-N-phenylhydroxylamine (3) was further latentiated by conversion to its O-acetyl (1a), O-methoxycarbonyl (1b), O-ethoxycarbonyl (1c), and O-methyl (2) derivatives. Similarly, pro-prodrug forms of nitroxyl were prepared by derivatization of the hydroxylamino moiety of methanesulfohydroxamic acid with N, O-bis-acetyl (7a), N,O-bis-methoxycarbonyl (7b), N, O-bis-ethoxycarbonyl (7c), and N-methoxycarbonyl-O-methyl (7d) groups. It was expected that the bioactivation of these prodrugs would initiate a cascade of nonenzymatic reactions leading to the ultimate liberation of NB or nitroxyl, thereby inhibiting AlDH. Indeed, the ester pro-prodrugs of both series were highly active in inhibiting yeast AlDH in vitro with IC50 values ranging from 21 to 64 microM. However, only 7d significantly raised ethanol-derived blood acetaldehyde levels when administered to rats, a reflection of the inhibition of hepatic mitochondrial AlDH-2.

Accumulation, depuration and hepatic mixed-function oxidase enzyme induction in juvenile rainbow trout and lake whitefish exposed to dietary 2,3,7,8-tetrachlorodibenzo -p-dioxin

Juvenile rainbow trout ( Oncorhynchus mykiss) and lake whitefish ( Coregonus clupeaformis) were exposed to three concentrations (40, 190, 400 pg g −1) of dietary 2,3,7,8-[ 3 H] tetrachlorodibenzo-p-dioxin (TCDD) to compare bioaccumulation and hepatic monooxygenase enzyme (MO) induction. Fish were exposed for 30 days followed by a 180 day depuration phase. Differences in the accumulation and depuration of TCDD were found between rainbow trout and lake whitefish, despite similar body size and lipid content. Assimilation efficiencies of TCDD were greater in lake whitefish (66–76%) than rainbow trout (43–58%), but TCDD half lives were shorter in lake whitefish (32–39 days) than in rainbow trout (73–83 days). Biomagnification factors (BMF) ranged from 1.6 to 1.8 in rainbow trout and from 0.8 to 0.9 in lake whitefish, confirming the known potential for biomagnification of TCDD in aquatic food webs. Reverse phase HPLC showed that a majority of the radioactivity in the rainbow trout bile was TCDD, with minor amounts present as a hydroxylated TCDD and as a glucuronide conjugate. MO enzyme induction, measured by ethoxyresorufin- O-deethylase (EROD), was observed in the rainbow trout after 10 days of exposure to 400 pg g −1 TCDD, and in the lake whitefish after 5 days of exposure to 380 pg g −1 TCDD. The whole fish threshold concentration for EROD induction by TCDD ranged between 15 and 45 pg g −1 (wet weight) for both species. EROD activity returned to control levels 120 and 80 days after the cessation of the treatments in the rainbow trout and lake whitefish, respectively. Growth rates were significantly reduced in trout and whitefish at whole fish concentrations (wet weight) of 150 ± 4.6 and 85 ± 8.3 pg g −1, respectively. Histological effects of the TCDD were found in the spleen and liver of the rainbow trout which had whole fish concentrations (wet weight) of 150 ± 4.6 pg mg −1and 72 ± 8.0 pg mg −1 TCDD, respectively.

Effect of sulfamethazine on phenobarbital and benzo[a]pyrene induced hepatic microsomal mixed function oxidase system in rats

Administration of sulfamethazine (300 mg/kg, i.p., single dose) to phenobarbital (80 mg/kg, i.p., 3 days) pretreated rats showed significant decrease in microsomal protein, electron transport components and drug metabolizing enzyme activities, compared with phenobarbital administration alone. Induction of mixed function oxidase enzymes due to phenobarbital was not affected by the pretreatment of sulfamethazine. Sulfamethazine administration to benzo[a]-pyrene (20 mg/kg, i.p., 2 days in oil) pretreated rats showed no significant change, but there was a slight decrease in cytochrome P450 and aminopyrine N-demethylase activity, compared with benzo[a]pyrene administration alone. A significant inhibition was observed in aminopyrine N-demethylase activity due to in vitro addition of sulfamethazine (3.5 mM) to microsomal incubations from untreated, sulfamethazine, phenobarbital and benzo[a]pyrene-treated rats. The results indicate that the phenobarbital induced cytochrome P450 is more susceptible to sulfamethazine than benzo[a]pyrene induced cytochrome P450.