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-'4C1-and (Z)-15- [15,16-3H2jtetracosenoic 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 02 and the inhibition by CO and the antibody to cytochrome P450 reductase strongly implicate the participation of a cytochrome P450 in this reaction.Long-chain hydrocarbons are abundant components in the cuticular lipids of plants and insects (1, 2), where they function to prevent water loss from the surface of the organism. In some insect species, including the housefly, Musca domestica, hydrocarbon components function as sex pheromones. The main component of the sex pheromone produced by the female housefly is (Z)-9-tricosene (Z9-23:Hy) (3), which functions as a short-range attractant and stimulant (4). In vertebrates, hydrocarbons function in the myelin sheath of peripheral nerves (5) and as components of uropygial gland secretions (6).The mechanism for hydrocarbon biosynthesis has proven to be elusive. Studies in the 1920s (7) suggested that two fatty acids condense head-to-head to form a ketone that is then reduced to the alkane. In an elegant series of experiments in the 1960s (reviewed in ref. -8), Kolattukudy and coworkers demonstrated that hydrocarbons are formed by the elongation of fatty acids, which are then converted to hydrocarbon by the loss of the carboxyl group, which was presumed to be a decarboxylation reaction (9,10). More recently, Kolattukudy and coworkers have obtained evidence from studies in a microorganism (11,12), a plant (13), a vertebrate (6), and an insect (14) that long-chain fatty acyl groups are reduced to aldehydes and then converted to hydrocarbons by a reductive decarbonylation mechanism. This mechanism does not require reduced pyridine nucleotides, and the carbonyl carbon is released as CO. In contrast, Gorgen and coworkers (15,16) presented evidence that 1-alkenes are formed by a decarboxylation mechanism in both plants and insects. We present evidence in this paper that hydrocarbon formation in the housefly occurs by the reduction of a long-chain acyl-CoA to an aldehyde that is then converted to the hydrocarbon and CO2 by a reaction that requires NADPH and 02 and involves cytochrome P450.

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.

The role of cytochrome P450 metabolism of fatty acids and lipid peroxidation in the alterations of the fatty acid composition of the liver and liver pathology was investigated. The CYP2E1 inhibitors partially prevented CYP2E1 induction by ethanol and completely blocked lipid peroxidation. However, the liver pathology induced by ethanol was only partially prevented as was the decrease in arachidonic acid in total liver lipid, triglycerides and cholesterol esters. This means that liver peroxidation induced by ethanol can not completely account for the liver pathology or the decrease in arachidonic acid caused by ethanol. Lauric acid omega-1 hydroxidation by the liver microsomes in vitro was increased by ethanol and partially blocked by CYP2E1 inhibitors. However, although ethanol feeding increased the total hydroxidation and epoxidation of arachidonic acid, these were not inhibited by CYP2E1 inhibitors. Thus the ethanol-induced arachidonic acid depletion is not likely due to CYP2E1 metabolism of arachidonic acid, since the severity of liver pathology correlated negatively with the decrease in arachidonic acid in the ethanol-fed rats. The increase in its metabolism by microsomes and decrease in synthesis may be an important mechanism of ethanol-induced liver injury.

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The growth rate of a human mammary carcinoma, MX-1, was significantly reduced in athymic "nude" mice fed fish oil. Tumors from the fish oil-fed animals also showed a greater sensitivity to two anti-neoplastic agents, mitomycin C and doxorubicin. Mitochondria were isolated from control livers, host livers and tumors from fish oil- and corn oil-fed animals, and increased levels of 20:5n-3 and 22:6n-3 were found in mitochondrial lipids in all three tissues from the fish oil-fed animals. To investigate the effect of dietary n-3 fatty acids on lipid metabolism, the activity of the acyl-CoA:carnitine acyltransferase and three acyl-CoA desaturases were measured. Carnitine acyltransferase activity toward all four acyl-CoA substrates tested was markedly increased in mitochondria from liver by feeding fish oil. In mitochondria from tumors, feeding fish oil resulted in an increased activity toward only 18:3n-3. These data suggest that fish oil may induce an increase in the oxidation of fatty acids. The delta 9-desaturase activity was decreased in microsomes from liver and tumor from fish oil-fed animals. However, both the delta 6 and delta 5 desaturases were increased in tumor and in control liver as a result of feeding fish oil. The delta 5 desaturase was not altered in microsomes from the host animals. The effect of fish oil on the delta 5 and delta 6 desaturases may involve alterations to metabolism of specific polyunsaturated fatty acids especially in the tumor tissue.