Proposed occupational exposure limits for select ethylene glycol ethers using PBPK models and Monte Carlo simulations.

Methoxyethanol (ethylene glycol monomethyl ether, EGME), ethoxyethanol (ethylene glycol monoethyl ether, EGEE), and ethoxyethyl acetate (ethylene glycol monoethyl ether acetate, EGEEA) are all developmental toxicants in laboratory animals. Due to the imprecise nature of the exposure data in epidemiology studies of these chemicals, we relied on human and animal pharmacokinetic data, as well as animal toxicity data, to derive 3 occupational exposure limits (OELs). Physiologically based pharmacokinetic (PBPK) models for EGME, EGEE, and EGEEA in pregnant rats and humans have been developed (M. L. Gargas et al., 2000, Toxicol. Appl. Pharmacol. 165, 53-62; M. L. Gargas et al., 2000, Toxicol. Appl. Pharmacol. 165, 63-73). These models were used to calculate estimated human-equivalent no adverse effect levels (NAELs), based upon internal concentrations in rats exposed to no observed effect levels (NOELs) for developmental toxicity. Estimated NAEL values of 25 ppm for EGEEA and EGEE and 12 ppm for EGME were derived using average values for physiological, thermodynamic, and metabolic parameters in the PBPK model. The uncertainties in the point estimates for the NOELs and NAELs were estimated from the distribution of internal dose estimates obtained by varying key parameter values over expected ranges and probability distributions. Key parameters were identified through sensitivity analysis. Distributions of the values of these parameters were sampled using Monte Carlo techniques and appropriate dose metrics calculated for 1600 parameter sets. The 95th percentile values were used to calculate interindividual pharmacokinetic uncertainty factors (UFs) to account for variability among humans (UF(h,pk)). These values of 1.8 for EGEEA/EGEE and 1.7 for EGME are less than the default value of 3 for this area of uncertainty. The estimated human equivalent NAELs were divided by UF(h,pk) and the default UFs for pharmacodynamic variability among animals and among humans to calculate the proposed OELs. This methodology indicates that OELs (8-h time-weighted average) that should protect workers from the most sensitive adverse effects of these chemicals are 2 ppm EGEEA and EGEE (11 mg/m(3) EGEEA, 7 mg/m(3) EGEE) and 0.9 ppm (3 mg/m(3)) EGME. These recommendations assume that dermal exposure will be minimal or nonexistent.

Physiological modeling reveals novel pharmacokinetic behavior for inhaled octamethylcyclotetrasiloxane in rats.

Octamethylcyclotetrasiloxane (D4) is an ingredient in selected consumer and precision cleaning products. Workplace inhalation exposures may occur in some D4 production operations. In this study, we analyzed tissue, plasma, and excreta time-course data following D4 inhalation in Fischer 344 rats (K. Plotzke et al., 2000, Drug Metab. Dispos. 28, 192-204) to assess the degree to which the disposition of D4 is similar to or different from that of volatile hydrocarbons that lack silicone substitution. We first applied a basic physiologically based pharmacokinetic (PBPK) model (J. C. Ramsey and M. E. Andersen, 1984, Toxicol. Appl. Pharmacol. 73, 159-175) to characterize the biological determinants of D4 kinetics. Parameter estimation techniques indicated an unusual set of characteristics, i.e., a low blood:air (P(b:a) congruent with 0.9) and a high fat:blood partition coefficient (P(f:b) congruent with 550). These parameters were then determined experimentally by equilibrating tissue or liquid samples with saturated atmospheres of D4. Consistent with the estimates from the time-course data, blood:air partition coefficients were small, ranging from 1.9 to 6.9 in six samples. Perirenal fat:air partition coefficients were large, from 1400 to 2500. The average P(f:b) was determined to be 485. This combination of partitioning characteristics leads to rapid exhalation of free D4 at the cessation of the inhalation exposure followed by a much slower redistribution of D4 from fat and tissue storage compartments. The basic PK model failed to describe D4 tissue kinetics in the postexposure period and had to be expanded by adding deep-tissue compartments in liver and lung, a mobile chylomicron-like lipid transport pool in blood, and a second fat compartment. Model parameters for the refined model were optimized using single-exposure data in male and female rats exposed at three concentrations: 7, 70, and 700 ppm. With inclusion of induction of D4 metabolism at 700 ppm (3-fold in males, 1-fold in females), the parameter set from the single exposures successfully predicted PK results from 14-day multiple exposures at 7 and 700 ppm. A common parameter set worked for both genders. Despite its very high lipophilicity, D4 does not show prolonged retention because of high hepatic and exhalation clearance. The high lipid solubility, low blood:air partition coefficient, and plasma lipid storage with D4 led to novel distributional characteristics not previously noted for inhaled organic hydrocarbons. These novel characteristics were only made apparent by analysis of the time-course data with PBPK modeling techniques.

The prevalence of insulin receptor antibodies in patients with systemic lupus erythematosus and related conditions.

Autoantibodies to the insulin receptor have been demonstrated to antagonize the physiologic actions of insulin, most often resulting in hyperglycemia unresponsive to massive doses of insulin (type B insulin resistance). Patients with these anti-insulin receptor antibodies typically have a coexistent autoimmune disorder, most commonly systemic lupus erythematosus (SLE) or undifferentiated autoimmune syndromes. Attempting to determine the prevalence and significance of anti-insulin receptor antibodies, sera from consecutive patients with SLE and early undifferentiated connective tissue disease (UCTD) were tested for the presence of anti-insulin receptor antibodies by radio-immuno assay. Thirty-eight patients participated in the study. Twenty-six had SLE and 12 had UCTD. One patient with SLE (2.6%) was positive for anti-insulin receptor antibodies. None of the patients demonstrated evidence of insulin resistance, hypoglycemia, ovarian hyperandrogenism, or acanthosis nigricans, findings commonly linked with the presence of anti-insulin receptor antibodies. The results presented here indicate that the incidence of anti-insulin receptor antibodies in patients with SLE or UCTD, without associated history of altered glucose metabolism, is quite low. Because in most cases the disturbance of glucose metabolism dominates the clinical picture at presentation and the associated systemic autoimmune syndrome presents either simultaneously with or subsequent to the diagnosis of diabetes, the measurement of anti-insulin receptor antibodies should be reserved for patients with indications of disordered glucose homeostasis.

Disposition of radioactivity in fischer 344 rats after single and multiple inhalation exposure to [(14)C]Octamethylcyclotetrasiloxane ([(14)C]D(4)).

The retention, distribution, metabolism, and excretion of [(14)C]octamethylcyclotetrasiloxane (D(4)) were studied in Fischer 344 rats after single and multiple exposures to 7, 70, or 700 ppm [(14)C]D(4). Subset groups were established for body burden, distribution, and elimination. Retention of inhaled D(4) was relatively low (5-6% of inhaled D(4)). Radioactivity derived from [(14)C]D(4) inhalation was widely distributed to tissues of the rat. Maximum concentrations of radioactivity in plasma and tissues (except fat) occurred at the end of exposure and up to 3 h postexposure. Maximum concentrations of radioactivity in fat occurred as late as 24 h postexposure. Fat was a depot, elimination of radioactivity from this tissue was much slower than from plasma and other tissues. With minor exceptions, there were no consistent gender effects on the distribution of radioactivity and the concentrations of radioactivity were nearly proportional to exposure concentration over the exposure range. Excretion of radioactivity was via exhaled breath and urine, and, to a much lesser extent, feces. Urinary metabolites included dimethylsilanediol and methylsilanetriol plus five minor metabolites. Relative abundance of these metabolites was the same from every test group. Elimination was rapid during the first 24 h after exposure and was slower thereafter (measured up to 168 h postexposure). In singly-exposed female (but not male) rats, small dose-dependent shifts in elimination pathways were seen. After multiple exposures, the elimination pathways were dose- and gender-independent. These data define possible pathways for metabolism of D(4) and allow estimation of the persistence of D(4) and/or its metabolites in rats.

Role of CYP2E1 in the pathogenesis of alcoholic liver disease: modifications by cAMP and ubiquitin-proteasome pathway.

The ethanol inducible isoform of cytochrome P450, CYP2E1, may play a role in ethanol-induced liver injury. Therefore, the factors which govern CYP2E1 degradation and turnover were investigated. These factors include cAMP, ubiquitin, proteasomal enzymes and CYP2E1 mRNA. Rats fed ethanol or pair-fed isocaloric dextrose were pair-fed with rats fed ethanol or dextrose treated with cAMP for 2 months. The liver pathology, regenerative activity, fatty acid composition, NFkappaB activation, ubiquitin conjugates and proteasomal enzymes were measured as were the apoprotein levels of CYP2E1, CYP3A, CYP4A and mRNA levels for CYP2E1 and ubiquitin expression. The results showed, that the cAMP treatment ameliorated the increase liver fat storage and changes in the fatty acid composition in the livers of ethanol fed rats. Other histologic features of alcoholic liver disease were not changed. Western blot quantitation showed that the amount of ubiquitin and ubiquitin conjugates were markedly reduced by ethanol treatment. Similarly, ethanol decreased the level of ubiquitin mRNA. cAMP ameliorated the inhibition of the proteasomal enzyme proteolysis caused by ethanol feeding. The ethanol-induced increase in the CYP2E1 protein was partially inhibited by cAMP treatment. cAMP treatment decreased CYP2E1 mRNA levels in both ethanol-fed and pair fed control rats. Likewise NFkappaB activation was not increased by ethanol but cAMP reduced the level of NFkappaB activation. CAMP treatment also reduced CYP4A but not CYP3A. The results support the concept that cAMP treatment partially protects the liver from ethanol-induced fatty liver by reducing CYP2E1 induction through cAMP's effects on CYP2E1 synthesis.

Technical and clinical aspects of spectrometric analysis of trace elements in clinical samples.

The capabilities of ICP-MS far exceed the slow, single-element analysis of GFAAS for determination of multiple trace elements. Additionally, its sensitivity is superior to that of DCP, ICP, and FAAS. The analytic procedure for ICP-MS is relatively straightforward and bypasses the need for digestion in many cases. It enables the physician to identify the target trace element(s) in intoxication cases, nutritional deficiency, or disease, thus eliminating the treatment delays experienced with sequential testing methods. This technology has its limitations as well. The ICP-MS cannot be used in the positive ion mode to analyze with sufficient sensitivity highly electronegative elements such as fluorine, because F+ is unstable and forms only by very high ionization energy. The ICP mass spectrometers used in most commercial laboratories utilize the quadrupole mass selector, which is limited by low resolution and, thus, by the various interferences previously discussed. For example, when an argon plasma is used, selenium (m/e 80) and chromium (m/e 52) in serum, plasma, and blood specimens are subject to polyatomic and molecular ion interferences. Low-resolution ICP mass spectrometers can therefore be used to analyze many trace elements, but they are not universal analyzers. High-resolution ICP-MS can resolve these interferences, but with greater expense. With the advent of more research and development of new techniques, some of these difficulties may be overcome, making this technique even more versatile. Contamination during sample collection and analysis causes falsely elevated results. Attention and care must be given to avoid contamination. Proper collection devices containing negligible amounts of trace elements should be used. Labware, preferably plastic and not glass, must be decontaminated prior to use by acid-washing and rinsed with [table: see text] de-ionized water. A complete description of sample collection and contamination has been written by Aitio and Jarvisalo as well as by Chan and Gerson. Lutz et al observed the ranges in blood shown in Table 4. We have adopted the ranges listed in Table 5 in urines of healthy, ambulatory, and community-dwelling individuals through a limited in-house study and review of literature. In conclusion, differentiation of trace element abnormalities (primary intoxication or disease versus secondary underlying disease) can be made only by utilizing results from trace element analyses in clinical specimens, medical history, and careful observation of symptoms. Repeat analysis on a second specimen collection is recommended when contamination is suspected.

Comparison of hexadecylphosphocholine with fish oil as an antitumor agent.

Hexadecylphosphocholine (HePC) reduced the growth of the human mammary tumor, MX-1, in the athymic nude mouse similar to the fish oil, MaxEPA. When used together, HePC and MaxEPA were additive towards reducing tumor growth. An unsaturated alkylphosphocholine mixture, ShisoPC, was not as effective as HePC in reducing tumor growth. MaxEPA reduced tumor PGE2 levels greater than 90%, while HePC and the ShisoPC only reduced tumor PGE2 40-60% with HePC being slightly better than ShisoPC. MaxEPA markedly increased the cellular omega 3 fatty acids and decreased 20:4 omega 6, the substrate for PGE2. HePC did not alter the tumor fatty acid composition, but it significantly lowered the total fatty acid concentration of the tumor by about 47%. In addition, phosphatidylcholine and sphingomyelin decreased in tumors from animals treated with HePC, and alterations in other phospholipids also were noted. These data suggest that different mechanisms exist for HePC and fish oil in reducing tumor growth.

Lipid peroxidation, CYP2E1 and arachidonic acid metabolism in alcoholic liver disease in rats.

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.

Effects of omega 3 fatty acids on receptor tyrosine kinase and PLC activities in EMT6 cells.

The effects of omega 3 fatty acids and epidermal growth factor (EGF) on the activity of receptor tyrosine kinase (RTK) and phospholipase C (phosphatidylinositol (PI)-specific PLC) were examined in EMT6 cells. The non-omega 3 treated, non-EGF stimulated cells served as controls. Treatment of the EMT6 cells with omega 3 fatty acids resulted in a 62% increase in RTK activity and a 67% increase in PI-specific PLC activity. When EGF was added to incubations for RTK activity, it stimulated the RTK activity 40% in the control cells and 130% in the omega 3-treated cells. When EGF was added to incubations for PI-specific PLC activity, a 54% increase in PI-specific PLC activity was observed in control cells and a 94% increase in the omega 3-treated cells. Thus, treating EMT6 cells with omega 3 fatty acids seems to increase RTK activity and PI-specific PLC activity to a similar extent, but has differential effects on the ability of these enzyme activities to be stimulated by EGF.

Using PBPK modeling and comprehensive realism methodology for the quantitative cancer risk assessment of butadiene.

The National Academy of Sciences and many others have noted the need for quantitative health risk assessment methodology that goes beyond a simple screening analysis based on upper bounds on risk. The Academy recommended adoption of methodologies which provide a higher-tier analysis based on realistic estimates of risk which reflect more of the available biological information. In recent years, scientists have challenged the assumption of low-dose linearity and other default assumptions in cancer risk assessment. These challenges have stimulated the continued evolution of quantitative risk assessment methodologies, because effective risk management requires accurate characterizations of uncertainty and greater utilization of cost-benefit analyses for decision making. "Comprehensive Realism" is an emerging quantitative weight-of-evidence based risk assessment methodology for both cancer and noncancer health effects which utilizes probability distributions and decision analysis techniques to reflect more of the available human and animal dose-response data. The current state of knowledge about the relative plausibility of alternative dose-response analyses is also addressed in this approach. The framework discussed here should lead to a higher-tier assessment of butadiene.

Predicting cancer risk from vinyl chloride exposure with a physiologically based pharmacokinetic model.

A physiologically based pharmacokinetic (PBPK) model capable of describing the metabolism of vinyl chloride (VC) in rats, mice, and humans has been developed and validated by comparison with experimental data from experiments not used in model development. This PBPK model has been used to predict measures of delivered dose (reactive VC metabolites produced in the livers of the affected species) hypothesized to be involved in the induction of liver angiosarcoma in rats, mice, and human populations exposed to VC. Measures of delivered dose in rats were fit to an empirical dose-response model (the linearized multistage model of Crump et al.) and used to make predictions of liver angiosarcoma incidence in mice and human populations exposed to VC. This procedure gave a good prediction of angiosarcoma incidence in mice. Predictions of angiosarcoma incidence in humans were more than two orders of magnitude lower than risk estimations which did not utilize pharmacokinetic data, but were still almost an order of magnitude higher than actually observed in exposed human populations.

In vivo and in vitro studies of perchloroethylene metabolism for physiologically based pharmacokinetic modeling in rats, mice, and humans.

In vivo experiments in rats and mice and in vitro experiments in rats, mice, and humans have been used to develop and validate a "2nd generation" physiologically based pharmacokinetic (PBPK) model for perchloroethylene (PERC). The refined PBPK model should be useful in the preparation of carcinogenic risk assessment based on amounts of PERC metabolites formed in the livers of rodents and humans according to procedures developed by EPA. A sensitivity analysis of the PBPK model revealed that the most significant uncertainties in this process (other than the choice of the appropriate dose/response model based on mechanism of action of PERC) were in the techniques used to estimate rates of PERC metabolism in humans. In vitro studies with human tissues reported help define what some have called the range of "equally reasonable alternatives" for estimating human risk.

Proposed mechanism for the cytochrome P450-catalyzed conversion of aldehydes to hydrocarbons in the house fly, Musca domestica.

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.

CYP-2E1 inhibitors partially ameliorate the changes in hepatic fatty acid composition induced in rats by chronic administration of ethanol and a high fat diet.

The objective of this study was to determine if ethanol-induced cytochrome P450 2E1 (CYP2E1) was responsible for the changes in hepatic fatty acids observed in rats fed ethanol intragastrically. We hypothesized that if CYP2E1 was responsible for these changes then CYP2E1 inhibitors fed with ethanol should prevent the ethanol-induced changes in fatty acids. We compared the fatty acid composition of the liver in rats fed ethanol alone with that in rats fed ethanol with the CYP2E1 inhibitors, diallyl sulfide and phenethyl isothiocyanate. In each experiment, rats pair-fed isocaloric glucose were included to determine the effect of the inhibitors alone on the hepatic fatty acid composition. The lobular distribution of succinic dehydrogenase was determined histochemically because the lobular distribution of CYP2E1 shifts to the periportal area in livers of rats fed CYP2E1 inhibitors. The CYP2E1 inhibitors ameliorated both the ethanol-induced changes in fatty acids and the shift in succinic dehydrogenase. Rats fed ethanol but no inhibitors had significantly greater hepatic total fatty acids and triglyceride fractions than when inhibitors were fed ethanol. Ethanol altered the fatty acid composition compared with rats fed ethanol with CYP2E1 inhibitors. The ratio of 20:4/18:2 was significantly lower and that of 18:1/18:0 was greater in alcohol-fed rats compared with their pair-fed controls. The CYP2E1 inhibitors inhibited many of the above effects of alcohol. The data suggest that the changes in the fatty acid composition due to ethanol ingestion are the result of CYP2E1-dependent lipid peroxidation and fatty acid metabolism.

Regulation of enzymatic activity involved in sex pheromone production in the housefly, Musca domestica.

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 KCl. 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 C27 and longer in the previtellogenic female to C23 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.

Structural basis of the phospholipid acyltransferase enzyme substrate specificity: a computer modeling study of the phospholipid acceptor molecule.

The activity of the 1-acyl-sn-glycero-3-phosphocholine acyltransferase enzyme (E.C. 2.3.1.??) was measured with three radically different acceptor substrates: 1-palmitoyl-sn-glycero-3-phosphocholine (P-sn-G3PC), 1-palmitoyl-sn-glycero-2-phosphocholine (P-sn-G2PC), and 1-hexadecyl-sn-glycero-3-phosphocholine (He-sn-G3PC). It was found that the enzyme had similar activity with P-sn-G3PC, the natural acceptor substrate, and with P-sn-G2PC. The enzyme showed no detectable activity toward He-sn-G3PC. These results are much different than would be expected from simple examination of the structures. Computer-assisted molecular modeling was done to study the geometrical configurations and to focus upon the similarities and differences of the three substrate acceptor molecules. Three bond distances were selected as important for enzyme recognition: the distance between the oxygen of the acceptor hydroxyl group and 1) the phosphorus; 2) the nitrogen; and 3) the oxygen bridge to the hydrocarbon chain. There were striking similarities for the bond distances of two of the three acceptor substrates, P-sn-G3PC and P-sn-G2PC. These were the two molecules that were shown to have activity with the enzyme. The bond distances found for the enzymically inactive acceptor substrate, He-sn-G3PC, differed significantly from P-sn-G3PC and P-sn-G2PC. Therefore, this latter molecule probably does not fit into the active site of the enzyme. The modeling data are also consistent with the experimental observation that He-sn-G3PC is not an inhibitor.

Unusual mechanism of hydrocarbon formation in the housefly: cytochrome P450 converts aldehyde to the sex pheromone component (Z)-9-tricosene and CO2.

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.

Phospholipid methylation in brain membrane preparations: kinetic mechanism.

The methylation reactions which convert phosphatidylethanolamine (PE) to phosphatidylcholine (PC) have been studied kinetically using exogenously added intermediates and crude membrane preparations from brain. The addition of exogenous PE resulted in no change in the methylation rates compared to that of endogenous PE. The addition of the two intermediates, monomethylphosphatidylethanolamine (PMME) and dimethylphosphatidylethanolamine (PDME), resulted in significantly increased rates of methylation and allowed the kinetic analysis of these latter two methylation reactions. The mechanism for this enzyme appears to be similar to human RBC (Reitz et al. (1989) J. Biol. Chem. 264, 8097-8106) which was a rapid-equilibrium random Bi-Bi sequential mechanism. There were some slight differences between the brain enzyme and that from the RBC, but there is little reason to suggest a fundamentally different mechanism. It is more likely that the differences may relate to an additional dead-end complex for the enzyme from brain such that saturation with AdoMet cannot eliminate AdoHcy inhibition. The KM values for the two phospholipid substrates were 41-44 microM and 39 microM for the methylation of PMME and PDME, respectively. The KM for S-adenosylmethionine (AdoMet) was 7-9 microM with PMME and 4 microM with PDME as the other substrates. The Ki(lipid) varied from 54 microM with PMME to 225 microM with PDME, and the Ki(AdoMet) was 11 microM with PMME and 21 microM with PDME. The product from the use of AdoMet, S-adenosylhomocysteine (AdoHcy), was shown to be a noncompetitive inhibitor of both lipid substrates as well as AdoMet. The methylation of PMME was somewhat higher in cerebellum and brain stem compared to cortex and striatum, but the methylation of PDME was similar in cerebellum, brain stem and cortex.

Dietary fatty acids and alcohol: effects on cellular membranes.

The consumption of ethanol has been shown to exert profound effects on cellular membranes which result in damage and/or adaptation. Both membrane lipids and proteins are affected, but because of the physicochemical properties of ethanol, many of the membrane effects are directly related to the interaction of ethanol with the lipid component of the membrane. In addition to the direct lipid-ethanol interaction, ethanol has been shown to dramatically alter lipid metabolism. Triacylglycerol accumulates dramatically in the liver, and biosynthesis of the polyunsaturated fatty acids seems to be altered via effects upon the acyl-CoA desaturases. Because precursors of both families of unsaturated fatty acids, i.e. omega 3 and omega 6 families, cannot be synthesized de novo, they must be supplied from dietary sources. Thus, the unsaturated membrane fatty acid composition depends upon these dietary fats and their metabolism via the desaturases. Further, the level of dietary fat seems to play a very important role in ethanol-induced damage to various cellular membranes. Diets with high levels of fat greatly enhance liver steatosis as well as liver membrane damage and liver fibrosis. By altering the composition of dietary fat to include either more saturated fatty acids, higher levels of a specific omega 6 fatty acid, alpha-linolenic acid, or higher levels of the omega 3 fatty acids, biochemical, physiological and neurobehavioral effects of ethanol have been shown to be modulated. Therefore, it appears that dietary fatty acids may play an important role in altering some of the deleterious effects of ethanol.