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A study of the substrate specificity and transfer of electrons for the conversion of very long fatty acyl-CoA to hydrocarbon and CO 2 by a cytochrome P 450 ( P 450hyd) type enzyme in microsomal preparations from abdominal epidermal tissue of the house fly, Musca domestica , showed that the enzyme which converts aldehyde to hydrocarbon contributes little to the chain length of the hydrocarbon products observed in vivo . NADPH (and not NADH) was required for the reduction of the fatty acyl-CoA to the corresponding aldehyde, and when both cofactors were added together, the amount of hydrocarbon formed from the acyl-CoA derivative was about the same as with NADPH alone. Both NADPH and NADH (less effectively) supported the conversion of the aldehyde to hydrocarbon and CO 2 by P450hyd. When both pyridine nucleotides were added together with the aldehyde substrate, conversion to hydrocarbon was intermediate between the levels formed with either cofactor alone, suggesting that the same electron carrier was transferring electrons from both NADH and NADPH and that cytochrome b 5 does not participate in the reaction. Antibody to house fly cytochrome P450 reductase inhibited the reaction when either NADPH or NADH was used. In addition, both NADP + and 2-AMP stimulated, rather than inhibited, hydrocarbon production in microsomes from female houseflies, presumably by inhibiting competing P450 enzymes which are supplied with electrons by the NADPH-cytochrome P450 reductase. These data suggest a novel mode of electron transfer to the P450hyd.

The mechanism of hydrocarbon biosynthesis was examined in the fleshfly Sarcophaga crassipalpis , the blowfly Phormia regina , the German cockroach Blattella germanica , the house cricket Acheta domesticus , the Mormon cricket Anabrus simplex , the dampwood termite Zootermopsis nevadensis and compared to the house fly, Musca domestica . Microsomal preparations from each species readily converted [9,10- 3 H 2 ]octadecanal (18:0 Ald) to heptadecane. NADPH and O 2 were required for enzymic activity in all cases, and very little hydrocarbon was formed under anaerobic conditions. Radio-GLC analyses of the head space gas formed from the metabolism of [1- 14 C]18:0 Ald by microsomes from M. domestica, S. crassipalpis, P. regina and B. germanica clearly showed that 14 CO 2 and not 14 CO was formed. Quantitation of the products from the metabolism of [1- 14 C] and [9,10- 3 H 2 ]18:0 Ald in microsomes from M. domestica, P. regina, S. crassipalpis, B. germanica and Z. nevadensis showed that an approximate 1 1 ratio of 14 CO 2 /[ 3 H] heptadecane was formed. The data support a mechanism in which the aldehyde is converted to hydrocarbon and CO 2 in insects.

Experiments were performed to elucidate the mechanism of hydrocarbon formation in microsomal preparations from the house fly, Musca domestica. Antibody to both house fly cytochrome P450 reductase and a purified cytochrome P450 (CYP6A1) from the house fly inhibited (Z)-9-tricosene (Z9-23:Hy) formation from [15,16-3H]-(Z)-15-tetracosenal (24:1 aldehyde). Chemical ionization-gas chromatography-mass spectrometry (CI-GC-MS) analyses of the n-tricosane formed by microsomal preparations from [2,2-2H2,2-13C]- and [3,3-2H2,3-13C]tetracosanoyl-CoA demonstrated that the deuteriums on the 2,2- and 3,3-positions were retained in the conversion to the hydrocarbon product. Likewise, CI-GC-MS analysis of the Z9-23:Hy formed from [1-2H]tetracosenal by microsomal preparations demonstrated that the aldehydic proton on the 1-carbon was transferred to the hydrocarbon product. Hydrogen peroxide, cumene hydroperoxide, and iodosobenzene were able to support hydrocarbon production from [3H]24:1 aldehyde in place of O2 and NADPH for short incubation times. From these data, a cytochrome P450 mechanism is proposed in which the perferryl iron-oxene, resulting from heterolytic cleavage of the O-O bond of the iron-peroxy intermediate, abstracts an electron from the C=O double bond of the carbonyl group of the aldehyde. The reduced perferryl attacks the 1-carbon of the aldehyde to form a thiyl-iron-hemiacetal diradical. The latter intermediate can fragment to form an alkyl radical and a thiyl-iron-formyl radical. The alkyl radical then abstracts the formyl hydrogen to produce the hydrocarbon and CO2.

Ovarian produced ecdysteroids regulate sex pheromone production in the female housefly, inducing the synthesis of (Z)-9-tricosene (Z9-23 : Hy), cis-9,10-epoxytricosane , (Z)-14-tricosen-10-one and methylalkanes. Experiments were performed to gain a detailed understanding of the processes affected by 20-hydroxyecdysone (20-HE) that result in sex pheromone production as the female becomes reproductively mature. A novel microsomal fatty acid synthetase (FAS) is present in the epidermal tissue and plays a role in producing the methyl-branched fatty acid precursors to the methylalkanes. This FAS is released from the microsomes in the presence of 3 M KC1. A major enzyme activity influenced by 20-HE is the fatty acyl-CoA elongation system. A shift in the chain length specificity of the products of the elongation system causes the change in the chain lengths of the alkenes produced to switch from C 27 , and longer in the previtellogenic female to C 23 , in the mature female. Data is presented indicating that it is the condensation activity of the elongation system that is affected. Z9-23:Hy arises from a 24 carbon acyl group which is reduced to an aldehyde, and then converted to the hydrocarbon. Data is presented demonstrating that it is the fatty acyl-CoA derivative and not the free fatty acid that is the substrate. There does not appear to be a chain length specificity which regulates the conversion of fatty acyl-CoAs to hydrocarbons as both 24 and 28 carbon fatty acyl-CoAs are converted to hydrocarbon by both males and females of all ages.

Many types of enzymes in living systems use hemin as a prosthetic group to catalyze oxidation/reduction reactions or for the binding/transport of reactive molecules (e.g. oxygen). For instance, several cytochromes of the mitochondrial electron transport chain are “heme” enzymes as are the major drug/xenobiotic-metabolizing enzymes of the endoplasmic reticulum, the cytochromes P450 (CYP or P450). The heme group of the P450s allows these enzymes to use redox chemistry to bind molecular oxygen and cleave the O-O bond, thus forming a reactive, high-valent oxygen species that can insert oxygen into otherwise stable carbon-hydrogen bonds of drugs/xenobiotics (White and Coon, 1980). The unfavorable thermodynamics of this type of reaction has caused the P450s to be likened to “catalytic blowtorches” (Schlichting et al., 2000), and the process is essential for the elimination and clearance of many lipophilic compounds ingested from the environment. Catalase is an important protective heme enzyme that is responsible for degrading N N

An unusual mechanism for hydrocarbon biosynthesis is proposed from work examining the formation of (Z)-9-tricosene (Z9-23:Hy), the major sex pheromone component of the female housefly, Musca domestica. Incubation of (Z)-15-[1-14C]- and (Z)-15-[15,16-3H2]tetracosenoic acid (24:1 fatty acid) with microsomes from houseflies gave equal amounts of [3H]Z9-23:Hy and 14CO2. The formation of CO2 and not CO, as reported for hydrocarbon formation in plants, animals, and microorganisms [Dennis, M. & Kolattukudy, P. E. (1992) Proc. Natl. Acad. Sci. USA 89, 5306-5310], was verified by trapping agents and by radio-GLC analysis. Incubation of (Z)-15-[15,16-3H2]tetracosenoyl-CoA with microsomal preparations in the presence of NADPH and O2 gave almost equal amounts of (Z)-15-3H2]tetrasosenal (24:1 aldehyde) and Z9-23:Hy. Addition of increasing amounts of hydroxylamine (aldehyde trapping agent) caused a decrease in hydrocarbon formation with a concomitant increase in oxime (aldehyde derivative) formation. The 24:1 aldehyde was efficiently converted to (Z)-9-tricosene only in the presence of both NADPH and O2. Bubbling carbon monoxide (20:80 CO/O2) or including an antibody against housefly cytochrome P450 reductase inhibited the formation Z9-23:Hy from 24:1 aldehyde. These data demonstrate an unusual mechanism for hydrocarbon formation in insects in which the acyl-CoA is reduced to the corresponding aldehyde and then carbon-1 is removed as CO2. The requirement for NADPH and O2 and the inhibition by CO and the antibody to cytochrome P450 reductase strongly implicate the participation of a cytochrome P450 in this reaction.

Eighteen methyl-branched fatty acids from the housfly, Musca domestica were identified by gas chromatography-mass spectrometry after reduction to the corresponding hydrocarbons. A deuterium was inserted on what was the carboxyl carbon and the straight chain components were removed by molecular sieve. The deuterium allowed the mass spectral determination of the methyl branch position with respect to the carboxyl end of the parent fatty acid methyl-branched fatty acids were characterized as n-2, n-3, n-4, n-5, n-6, n-7, n-8, and n-9 monomethyl fatty acids of 15—19 carbons and a n-3, 7 dimethyl fatty acid of 18 total carbons. These methyl-branched fatty acids have similar branching patterns and are presumed to the precursors to the methyl-branched hydrocarbons, some of which function as an arrestant in the female sex pheromone. With increasing concentrations of methylmalonyl-CoA, its incorporation into methyl-branched fatty acids increased in both day 1 and day 4 males and females using both microsomal and soluble fatty acid synthases (FAS). Methylmalonyl-CoA inhibited both the soluble and microsomal FAS in a competitive manner. The data on the incorporation of methylmalonyl-CoA into methyl-branched fatty acids by day 1 and day 4 males and females indicate that the regulation of methyl-branched hydrocarbon synthesis does not reside at the level of fatty acid synthesis, but must occur during the process of the fatty acyl-CoA elongation or reductive conversion of very long chain fatty acyl-CoAs to hydrocarbons.

The goal of this study was to characterize the effects of CYP1A2·CYP2B4 complex formation on the rates and efficiency of toluene metabolism by comparing the results from simple reconstituted systems containing P450 reductase (CPR) and a single P450 to those using a mixed system containing CPR and both P450s. In the mixed system, the rates of formation of CYP2B4-specific benzyl alcohol and p-cresol were inhibited, whereas that of CYP1A2-specific o-cresol was increased, results consistent with the formation of a CYP1A2·CYP2B4 complex in which the CYP1A2 moiety has a higher affinity for CPR binding. Comparison of the rates of NADPH oxidation and production of hydrogen peroxide and excess water by the simple and mixed systems indicated that excess water formed at a much lower rate in the mixed system. The commensurate increase in the rate of CYP1A2-specific product formation suggested the P450·P450 interaction increased the rate of the putative rate-limiting step of CYP1A2 catalysis, abstraction of a hydrogen radical from the substrate. Cumene hydroperoxide-supported metabolism was measured to determine whether the effects of the P450·P450 interaction required the presence of CPR. Peroxidative metabolism was not affected by the interaction of the two P450s, even with CPR present. However, CPR did stimulate peroxidative metabolism by the simple system containing CYP1A2. These results suggest the major functional effects of the P450·P450 interaction are mediated by changes in the relative abilities of the P450s to receive electrons from CPR. Furthermore, CPR may play an effector role by causing a conformational change in CYP1A2 that makes its metabolism more efficient.

Cytochrome P450 is part of an electron transport chain found in the endoplasmic reticulum (ER), with its catalytic function requiring interactions with NADPH-cytochrome P450 reductase (CPR). The goals of this study were to examine how the P450 system proteins are organized in the membrane and to determine whether they are distributed in detergent-resistant lipid microdomains (DRM). Isolated liver microsomes from untreated rabbits were treated with 1% Brij 98, and DRMs were isolated via sucrose gradient centrifugation. Lipid analysis showed that DRM fractions were enriched in cholesterol and sphingomyelin, similar to that found with plasma membrane DRMs. Approximately 73% of CYP1A2 and 68% of CPR resided in DRM fractions, compared with only 33% of total ER proteins. These DRMs were found to be cholesterol-dependent: CPR and CYP1A2 migrated to the more dense regions of the sucrose gradient after cholesterol depletion. CYP1A2 function was studied in three purified lipid vesicles consisting of 1) phosphatidylcholine (V-PC), 2) lipids with a composition similar to ER lipids (V-ER), and 3) lipids with a composition similar to the DRM fractions (V-DRM). Each system showed similar substrate binding characteristics. However, when the association between CPR and CYP1A2 was measured, V-ER and V-DRM liposomes produced lower apparent Km values compared with V-PC without any significant change in Vmax. These findings suggest that CYP1A2 and CPR reside in ER-DRMs and that the unique lipid components of these domains enhance CYP1A2 substrate metabolism through greater efficiency in CPR-CYP1A2 binding.

Although lipids are essential for the optimal activity of the cytochromes P450 monooxygenase system, relatively little is known about the membrane environment in which these enzymes function. One approach used to mimic the structural arrangement of lipids and enzymes within the endoplasmic reticulum is to physically incorporate the cytochromes P450 and their redox partners in a vesicle bilayer of phospholipids. Several methods have been devised for this purpose. This chapter describes a method in which the P450 monooxygenase system is incorporated by first, solubilizing the enzymes and lipid with sodium glycocholate. After the protein and lipid aggregates are dispersed, the detergent is removed by adsorption using BioBeads SM-2 resin which leads to the formation of bilayer vesicles of phospholipid containing incorporated cytochrome P450 and NADPH cytochrome P450 reductase. This procedure requires relatively a short preparation time, provides concentrated reconstituted systems that can be used in a wide range of applications, allows for several enzyme samples to be prepared simultaneously so that different conditions can be compared, and results in minimal loss of active enzyme.

In a previous study from our laboratory (Drug Metab Dispos 34: 660-666, 2006), we found several limitations with published methods (cholate gel filtration and cholate dialysis) for the incorporation of cytochromes P450 and P450 reductase into phospholipid vesicles. We found that a significant proportion of reductase was not incorporated in the vesicles when the amount of reductase was equal to or greater than that of CYP2B4 in the systems reconstituted with phosphatidylcholine. Furthermore, implementation of these methods compromised the ability of the CYP2B4 to form a ferrous carbon monoxy complex. In the current study, a comparison of results using the detergent-dialysis method with five similar detergents having the "bile salt" ring structure showed that glycocholate results in the greatest incorporation of reductase and the least loss in the ferrous carbon monoxy CYP2B4 complex. The method is further improved by using Bio-Beads SM-2 to remove detergent instead of the lengthy dialysis procedure or size exclusion chromatography that significantly dilutes the protein and lipid concentrations of the preparation. The method is shown to be applicable over a range of lipid/CYP2B4 ratios, and by using assay methods for total lipid, reductase, and CYP2B4, this improved reconstitution method resulted in increased incorporation efficiencies while minimizing the protein degradation inherent with these procedures.

2-Ethynylnaphthalene (2EN) is a mechanism-based inhibitor of CYP2B4 with two components to the inhibition, (1) enzyme inactivation, which requires covalent binding of the 2EN metabolite, and (2) reversible inhibition by 2EN itself. Both inhibitory components were examined using several different CYP2B4 substrates. Preincubation of CYP2B4 with 2EN led to a time-dependent inactivation of each of the CYP2B4-dependent activities examined; however, the ability of 2EN to reversibly inhibit CYP2B4 depended on the substrate employed, which is inconsistent with classical inhibition patterns. The degree 2EN’s reversible inhibition was shown not to correlate with the substrate affinity for the active site, but with parameters related to the molecular size of the substrate. The results are consistent with 2EN and the smaller substrates simultaneously fitting in the CYP2B4 active site, leading to very little inhibition. Larger substrates exhibited greater degrees of inhibition because of their inability to co-bind with inhibitor in the active site.

A species difference was observed in the excretion pathway of 2-[[5,7-dipropyl-3-(trifluoromethyl)-1,2-benzisoxazol-6-yl]oxy]-2-methylpropanoic acid (MRL-C), an alpha-weighted dual peroxisome proliferator-activated receptor alpha/gamma agonist. After intravenous or oral administration of [14C]MRL-C to rats and dogs, radioactivity was excreted mainly into the bile as the acyl glucuronide metabolite of the parent compound. In contrast, when [14C]MRL-C was administered to monkeys, radioactivity was excreted into both the bile and the urine as the acyl glucuronide metabolite, together with several oxidative metabolites and their ether or acyl glucuronides. Incubations in hepatocytes from rats, dogs, monkeys, and humans showed the formation of the acyl glucuronide of the parent compound as the major metabolite in all species. The acyl glucuronide and several hydroxylated products, some which were glucuronidated at the carboxylic acid moiety, were observed in incubations of MRL-C with NADPH- and uridine 5'-diphosphoglucuronic acid-fortified liver microsomes. However, metabolism was more extensive in the monkey microsomes than in those from the other species. When the acyl glucuronide metabolite of MRL-C was incubated with NADPH-fortified liver microsomes, in the presence of saccharo-1,4-lactone, it underwent extensive oxidative metabolism in the monkey but considerably less in the rat, dog, and human liver microsomes. Collectively, these data suggested that the oxidative metabolism of the acyl glucuronide might have contributed to the observed in vivo species differences in the metabolism and excretion of MRL-C.

A species difference was observed in the excretion pathway of 2-[[5,7-dipropyl-3-(trifluoromethyl)-1,2-benzisoxazol-6-yl]oxy]-2-methylpropanoic acid (MRL-C), an alpha-weighted dual peroxisome proliferator-activated receptor alpha/gamma agonist. After intravenous or oral administration of [14C]MRL-C to rats and dogs, radioactivity was excreted mainly into the bile as the acyl glucuronide metabolite of the parent compound. In contrast, when [14C]MRL-C was administered to monkeys, radioactivity was excreted into both the bile and the urine as the acyl glucuronide metabolite, together with several oxidative metabolites and their ether or acyl glucuronides. Incubations in hepatocytes from rats, dogs, monkeys, and humans showed the formation of the acyl glucuronide of the parent compound as the major metabolite in all species. The acyl glucuronide and several hydroxylated products, some which were glucuronidated at the carboxylic acid moiety, were observed in incubations of MRL-C with NADPH- and uridine 5'-diphosphoglucuronic acid-fortified liver microsomes. However, metabolism was more extensive in the monkey microsomes than in those from the other species. When the acyl glucuronide metabolite of MRL-C was incubated with NADPH-fortified liver microsomes, in the presence of saccharo-1,4-lactone, it underwent extensive oxidative metabolism in the monkey but considerably less in the rat, dog, and human liver microsomes. Collectively, these data suggested that the oxidative metabolism of the acyl glucuronide might have contributed to the observed in vivo species differences in the metabolism and excretion of MRL-C.

The pre-steady-state reduction of cytochrome P450 (P450) 2B4 by P450 reductase (reductase) was modeled by assuming that an equilibrium between three catalytic conformers of P450 regulates the multi-phasic reduction of the enzyme. This model was compared to a model of reduction involving a minimum number of phases. Based on several criteria, the former model seems to provide an improved fit to the reduction data. Substrates were divided into two groups based on their effects at different concentrations of reductase. Surprisingly, in the presence of some substrates (group 1) but not others (group 2), the rate of reduction was actually slower with an excess of reductase than with equimolar reductase and P450. Presumably, oxidized reductase binds differently to P450 than reduced reductase. A schematic model based on two sites of interaction between reductase and P450 2B4 is offered to explain the unusual reduction kinetics with the two different groups of substrates.

Microwave irradiation (960-MHz CW) at a specific absorption rate (SAR) near 1.5 W/kg induced bradycardia in isolated rat heart as reported earlier. When parasympathetic and sympathetic nerves were simultaneously blocked, respectively, by atropine and by propranolol, no significant effect of irradiation was observed. Previous experiments have revealed that atropine plus irradiation produces tachycardia and propranolol plus irradiation produces bradycardia. The results may indicate a microwave neuron or microwave-synapse interaction by a mechanism other than generalized heating of tissues.

To study the renal hemodynamic response to a large intravenous bolus of a radiocontrast agent, 8 ml/kg body weight of 60% diatrizoate meglumine (D-60) was infused over 30 seconds in both normal rats and rats with streptozotocin-induced diabetes. The effect of equiosmolar mannitol (1350 mOsm/kg) was compared with the D-60 response in normal rats to examine the potential role of hypertonicity in mediating a response. A similar two-phase response was seen in all three groups. The effect of D-60 in normal rats was similar to that of mannitol, but all responses were reduced in diabetic rats. During Phase I in normal rats in response to D-60 there was a transient three-minute reduction in blood pressure (BP), and renal blood flow (RBF) fell by 62 +/- 7%. During Phase II blood pressure did not change from normal baseline values, but RBF fell by 29 +/- 5%; GFR fell by 42 +/- 3%, and filtration fraction (FF) diminished. In diabetic rats baseline FF was lower than normal and was not further reduced after infusion of D-60. It is suggested that D-60 reduces RBF and GFR by a nonspecific osmotic effect, perhaps related to tubuloglomerular feedback or to an acute increase in intratubular pressure. Responses to these mechanisms may be reduced in diabetic rats with a chronic glycosuric osmotic diuresis.

Alarm substances and fright reactions were found in three species of Cyprinidae from the southeastern United States (Notropis venustus, N. texanus and Hybopsis aestivalis). Three types of fright reactions were observed corresponding to the vertical distribution of the test species in the natural habitat (top-water, mid-water and bottom). Evidence was found that Gambusia affinis and Fundulus olivaceus (Poeciliidae and Cyprinodontidae) respond to skin extract from their own species with a reaction that may be comparable to the fright reaction known in the Ostariophysi and Gonorynchiformes. A predator odor capable of eliciting a fright response in local prey species was found in three North American predatory fishes \[Lepomis macrochirus, and Micropterus punctulatus (Centrarchidae); Esox niger (Esocidae)\] and in two South American fishes, Astronotus ocellatus and Cichlasoma severum (Cichlidae). The reactions to predator odor by the prey were similar in appearance to those observed in experiments us...

Black bullheads were able to accumulate 60Co from water. During uptake the gills contained from 19% to 30% of the whole-body 60Co activity. After 3 days of 60Co uptake the flesh accounted for 16% of the total activity. Fish that were dosed in a single feeding lost 95.5% of the initial activity in one day of excretion whereas fish that were exposed in 60Co-dosed water lost 28% of the initial activity during the first day of excretion. Elimination of 60Co after intake from water or from food occurred in three components. The initial rapid loss of activity after uptake from food had a biological half-life of 1.5 days, compared to 3.4 days for the initial component after uptake via water. The intermediate components had biological half-lives of 35 and 40.5 days, respectively, after water uptake and after food uptake. The third components in both cases had long, undetermined biological half-lives. Radiocobalt and stable cobalt analyses showed that the blood and blood-rich organs, particularly the kidney, were principal sites of cobalt concentration.

The poor correlation between clinical salicylate toxicity and serum blood levels is reapproached in light of recent evidence linking clinical severity with initial volume of distribution (Vd). It is recognized that two variables alter salicylate Vd in such manner that serum salicylate levels are misleading (thus, the change in Vd is not detected by present methods). These variables are serum protein binding and the pH-dependent ionized/un-ionized ratio in the unbound salicylate fraction. Measurements of salicylate concentration in the cerebro spinal fluid (CSF) would circumvent these variables, but would be clinically impractical. Thus, an alternative is sought to the inexact total serum salicylate levels and the impractical CSF salicylate levels for assessment of the severity of salicylate poisoning. This study indicates that, in dogs, serum unbound salicylate levels closely reflect CSF salicylate levels, even as a decrease in serum protein binding is in progress. However, serum unbound salicylate concentration does not reflect CSF salicylate concentration as a decrease in serum pH is elicited (CSF salicylate actually increased as serum unbound salicylate decreased). On the other hand, serum unbound salicylate measurement would seem preferable to total serum salicylate measurements now used in that the total value decreased markedly as either protein binding change or acidosis produced a change in distribution and the resultant increase in CSF salicylate.