Role of Cell Death in Toxicology: Does It Matter How Cells Die?

My research activity started with studies on drug metabolism in rat liver microsomes in the early 1960s. The CO-binding pigment (cytochrome P450) had been discovered a few years earlier and was subsequently found to be involved in steroid hydroxylation in adrenal cortex microsomes. Our early studies suggested that it also participated in the oxidative demethylation of drugs catalyzed by liver microsomes, and that prior treatment of the animals with phenobarbital caused increased levels of the hemoprotein in the liver, and similarly enhanced rates of drug metabolism. Subsequent studies of cytochrome P450-mediated metabolism of toxic drugs in freshly isolated rat hepatocytes characterized critical cellular defense systems and identified mechanisms by which accumulating toxic metabolites could damage and kill the cells. These studies revealed that multiple types of cell death could result from the toxic injury, and that it is important to know which type of cell death results from the toxic injury.

HLM-beads: Rapid Assessment of Nonspecific Binding to Human Liver Microsomes Using Magnetizable Beads.

In early drug development, drug-drug interaction risk is routinely assessed using human liver microsomes (HLMs). Nonspecific binding of drugs to HLMs can affect the determination of accurate enzyme parameters (Km, Ki, KI). Previously, we described a novel in vitro model consisting of HLMs bound to magnetizable beads [HLM-magnetizable-beads system (HLM-beads)]. The HLM-beads enable rapid separation of HLMs from incubation media by applying a magnetic field. Here, HLM-beads were further characterized and evaluated as a tool to assess HLM nonspecific binding of small molecules. The free fractions (fu,mic) of 13 compounds (chosen based on their pKa values) were determined using HLM-beads under three HLM concentrations (0.025, 0.50, and 1.0 mg/ml) and compared with those determined by equilibrium dialysis. Most fu,mic values obtained using HLM-beads were within 0.5- to 2-fold of the values determined using equilibrium dialysis. The highest fold difference were observed for high binders itraconazole and BIRT2584 (1.9- to 2.9-fold), as the pronounced adsorption of these compounds to the equilibrium dialysis apparatus interfered with their fu,mic determination. Correlation and linear regression analysis of the fu,mic values generated using HLM-beads and equilibrium dialysis was conducted. Overall, a good correlation of fu,mic values obtained by the two methods were observed, as the r and R2 values from correlational analysis and linear regression analysis were >0.9 and >0.89, respectively. These studies demonstrate that HLM-beads can produce comparable fu,mic values as determined by equilibrium dialysis while reducing the time required for this type of study from hours to only 10 minutes and compound apparatus adsorption. SIGNIFICANCE STATEMENT: This work introduces a new method of rapidly assessing nonspecific microsomal binding using human liver microsomes bound to magnetizable beads.

In vitro-in vivo extrapolation of metabolic clearance using human liver microsomes: factors showing variability and their normalization.

In vitro-in vivo extrapolation (IVIVE) using human liver microsomes has been widely used to predict metabolic clearance, but some of the factors used in the process of prediction show variability for the same compound: notably, microsomal intrinsic clearance values corrected by the unbound fraction (CLint, u), physiological parameters used for scale-up, and the source of in vivo clearance data.The purpose of this study was to assess the correlation between in vitro and in vivo CLint with a focus on factors showing variability using four cytochrome P450 (CYP)3A substrates.We surveyed in vivo clearance values in literature and also determined the microsomal CLint, u values. A scaling factor (SFdirect) was defined as in vivo CLint divided by the microsomal CLint, u, which ranged from 1190 to 2310 (mg protein per kg body weight). The application of a mean SFdirect of 1600 (mg protein per kg body weight) and further normalization by the microsomal CLint, u values of midazolam, the most commonly used substrate, resulted in improved prediction accuracy for CLint, u values from various microsomal batches.The results suggest the normalization of variability might be useful for predicting the in vivo CLint.

Single-Nucleotide Polymorphisms in Cytochrome P450 2E1 (CYP2E1) 3'-Untranslated Region Affect the Regulation of CYP2E1 by miR-570.

Human cytochrome P450 2E1 (CYP2E1) catalyzes the metabolism of numerous xenobiotics, including acetaminophen and ethanol. CYP2E1 expression is known to be extensively regulated by post-transcriptional and post-translational mechanisms. A previous study had reported that a single-nucleotide polymorphism (SNP) 1561A>G in the 3'-untranslated region (3'-UTR) of CYP2E1 leads to a decreased CYP2E1 mRNA level in human peripheral blood mononuclear cells. In this study, we examined the possibility that microRNA(s) (miR) may be involved in the SNP-mediated modulation of CYP2E1 expression. Genotyping and sequencing analyses revealed that another SNP, 1556T>A, in the 3'-UTR was in complete linkage disequilibrium with the SNP 1561A>G. We termed the alleles with 1556T and 1561A or 1556A and 1561G haplotype I or II, respectively. A luciferase assay revealed that miR-570 recognizes the CYP2E1 3'-UTR of haplotype I but not haplotype II. Human embryonic kidney 293 (HEK293) cell lines stably expressing human CYP2E1 that included the 3'-UTR of haplotype I or II (HEK/2E1(I) or HEK/2E1(II) cells, respectively) were established. Overexpression of miR-570 significantly decreased the CYP2E1 protein level in the HEK/2E1(I) cells but not in the HEK/2E1(II) cells. In seven human livers with diplotype I/I, the CYP2E1 protein levels were inversely correlated with the miR-570 levels, but no relationship was observed in 25 human livers with diplotypes I/II and II/II. Collectively, it was demonstrated that human CYP2E1 was regulated by miR-570 in a genotype-dependent manner. This report describes the first proof that SNP in 3'-UTR of human P450 affects binding of miRNA to modulate the expression in the liver.

Dibenzazepines and dibenzoxazepines as sodium channel blockers.

We have identified two related series of dibenzazepine and dibenzoxazepine sodium channel blockers, which showed good potency on Nav1.7 in FLIPR-based and electrophysiological functional assays.

Pharmacokinetics and drug interactions of eslicarbazepine acetate.

Eslicarbazepine acetate (ESL) is a novel once-daily antiepileptic drug (AED) approved in Europe since 2009 that was found to be efficacious and well tolerated in a phase III clinical program in adult patients with partial onset seizures previously not controlled with treatment with one to three AEDs, including carbamazepine (CBZ). ESL shares with CBZ and oxcarbazepine (OXC) the dibenzazepine nucleus bearing the 5-carboxamide substitute, but is structurally different at the 10,11 position. This molecular variation results in differences in metabolism, preventing the formation of toxic epoxide metabolites such as carbamazepine-10,11-epoxide. Unlike OXC, which is metabolized to both eslicarbazepine and (R)-licarbazepine, ESL is extensively converted to eslicarbazepine. The systemic exposure to eslicarbazepine after ESL oral administration is approximately 94% of the parent dose, with minimal exposure to (R)-licarbazepine and OXC. After ESL oral administration, the effective half-life (t(1/2,eff) ) of eslicarbazepine was 20-24 h, which is approximately two times longer than its terminal half-life (t(1/2)). At clinically relevant doses (400-1,600 mg/day) ESL has linear pharmacokinetics (PK) with no effects of gender or moderate liver impairment. However, because eslicarbazepine is eliminated primarily (66%) by renal excretion, dose adjustment is recommended for patients with renal impairment. Eslicarbazepine clearance is induced by phenobarbital, phenytoin, and CBZ and it dose-dependently decreases plasma exposure of oral contraceptive and simvastatin.

Discovery and SAR of small molecule PAR1 antagonists.

High-throughput screening resulted in the identification of a small molecule inhibitor of PAR1. Optimisation of the initial hit led to the discovery of compounds 34 and 49, which displayed antithrombotic activity in an arteriovenous shunt model in the rat after iv administration.

Utilizing Pediatric Physiologically Based Pharmacokinetic Models to Examine Factors That Contribute to Methadone Pharmacokinetic Variability in Neonatal Abstinence Syndrome Patients.

Chronic intrauterine exposure to psychoactive drugs often results in neonatal abstinence syndrome (NAS). NAS is the symptomatic drug withdrawal in newborns that generally occurs after in utero chronic opioid exposure. Methadone is an opioid analgesic commonly prescribed for pharmacologic management of NAS. It exhibits high pharmacokinetic (PK) variability. The current study used physiologically based PK modeling to predict the PK profile of methadone in 20 newborns treated for NAS. The physiologically based PK simulations adequately predicted the PK profile of the clinical data for 45% of the patients. Sensitivity analyses were conducted to explore contributing factors to methadone PK variability. The data suggest that P450 enzymatic activity impacts the clearance of methadone in virtual adults and neonates, while the contribution of cardiac output may be negligible. Understanding maturational and/or pharmacogenetic changes in cytochrome P450 enzymatic activity may further explain the large PK variability of methadone in newborns with NAS and will help individualized treatment.

A heterotrimeric G protein, G alpha i-3, on Golgi membranes regulates the secretion of a heparan sulfate proteoglycan in LLC-PK1 epithelial cells.

A heterotrimeric G alpha i subunit, alpha i-3, is localized on Golgi membranes in LLC-PK1 and NRK epithelial cells where it colocalizes with mannosidase II by immunofluorescence. The alpha i-3 was found to be localized on the cytoplasmic face of Golgi cisternae and it was distributed across the whole Golgi stack. The alpha i-3 subunit is found on isolated rat liver Golgi membranes by Western blotting and G alpha i-3 on the Golgi apparatus is ADP ribosylated by pertussis toxin. LLC-PK1 cells were stably transfected with G alpha i-3 on an MT-1, inducible promoter in order to overexpress alpha i-3 on Golgi membranes. The intracellular processing and constitutive secretion of the basement membrane heparan sulfate proteoglycan (HSPG) was measured in LLC-PK1 cells. Overexpression of alpha i-3 on Golgi membranes in transfected cells retarded the secretion of HSPG and accumulated precursors in the medial-trans-Golgi. This effect was reversed by treatment of cells with pertussis toxin which results in ADP-ribosylation and functional uncoupling of G alpha i-3 on Golgi membranes. These results provide evidence for a novel role for the pertussis toxin sensitive G alpha i-3 protein in Golgi trafficking of a constitutively secreted protein in epithelial cells.

Cargo selection by the COPII budding machinery during export from the ER.

Cargo is selectively exported from the ER in COPII vesicles. To analyze the role of COPII in selective transport from the ER, we have purified components of the mammalian COPII complex from rat liver cytosol and then analyzed their role in cargo selection and ER export. The purified mammalian Sec23-24 complex is composed of an 85-kD (Sec23) protein and a 120-kD (Sec24) protein. Although the Sec23-24 complex or the monomeric Sec23 subunit were found to be the minimal cytosolic components recruited to membranes after the activation of Sar1, the addition of the mammalian Sec13-31 complex is required to complete budding. To define possible protein interactions between cargo and coat components, we recruited either glutathione-S-transferase (GST)-tagged Sar1 or GST- Sec23 to ER microsomes. Subsequently, we solubilized and reisolated the tagged subunits using glutathione-Sepharose beads to probe for interactions with cargo. We find that activated Sar1 in combination with either Sec23 or the Sec23-24 complex is necessary and sufficient to recover with high efficiency the type 1 transmembrane cargo protein vesicular stomatitis virus glycoprotein in a detergent-soluble prebudding protein complex that excludes ER resident proteins. Supplementing these minimal cargo recruitment conditions with the mammalian Sec13-31 complex leads to export of the selected cargo into COPII vesicles. The ability of cargo to interact with a partial COPII coat demonstrates that these proteins initiate cargo sorting on the ER membrane before budding and establishes the role of GTPase-dependent coat recruitment in cargo selection.

Fluidity of the rat liver microsomal membrane: increase at birth.

The lipid apparent microviscosity of the rat liver microsomal membrane on the first day after birth was found to be half of that observed on the last day of fetal life. This remarkable perinatal fluidization of the membrane resulted from a marked increase in the molar ratio of phospholipids to cholesterol.

Downregulation of the male-specific hepatic microsomal steroid 16 alpha-hydroxylase, cytochrome P-450UT-A, in rats with portal bypass. Relevance to estradiol accumulation and impaired drug metabolism in hepatic cirrhosis.

Elevated serum estradiol concentrations and specific changes in the biliary excretion of some androstenedione metabolites have been reported in male rats with portal bypass produced by portal vein ligation (PVL). In this study, the hypothesis that male-specific forms of cytochrome P-450 are altered after PVL was tested by measuring microsomal steroid hydroxylase activities. Consistent with earlier findings in the intact animal, androstenedione 16 alpha-hydroxylase activity was reduced after PVL to 44% of control (P less than 0.05). Other pathways of androstenedione hydroxylation, and total estrogen formation (after androstenedione aromatization) were unchanged. Although total estrogen formation was not different, a sevenfold greater proportion of estradiol was produced in PVL rat microsomes. Additional experiments revealed that PVL selectively reduced the rate of microsomal estradiol 16 alpha-hydroxylation (to 56% of control, P less than 0.02). Levels of cytochrome P-450UT-A, the microsomal steroid 16 alpha-hydroxylase, were lower after PVL (56% of control, P less than 0.05), so that the present observations are consistent with the earlier suggestion that portal bypass is associated with the selective downregulation of this enzyme. Since downregulation of cytochrome P-450UT-A also occurs in experimental hepatic cirrhosis, portal hypertension may well contribute significantly to altered drug metabolism in liver disease. Impaired hepatic elimination of androstenedione by hydroxylation may indirectly enhance extrahepatic aromatization of the androgen. The decreased activity of hepatic estradiol 16 alpha-hydroxylation after PVL would enhance the accumulation of estradiol, the biologically more potent estrogen.

Harmonised high throughput microsomal stability assay.

INTRODUCTION: Prediction of human pharmacokinetics from in vitro assays and pre-clinical data is an integral part of drug discovery. In vitro stability metabolic studies can provide an estimate of in vivo hepatic intrinsic clearance through inclusion of biological scaling factors. Many labs have personalised stability protocols including marker compounds and have adopted QC criteria and assay limits to ensure data integrity. Contract research organisations (CRO's) provide integrated drug discovery support to academic and pharmaceutical/biotechnology institutions to progress their in-house projects. The majority of these clients have established in-house protocols with associated criteria to ensure data consistency between in-house and external labs. METHODS: In this study, numerous assay variables were condensed into one harmonised assay format and a range of compounds with diverse physicochemical properties were evaluated. The protocols were diverse with respect to the following attributes: buffer, microsomal concentration and species strain. RESULTS: Comparison of human lots in vitro CLint between the traditional and consolidated assay formats showed a good correlation with no significant difference. A clear relationship was demonstrated between strains. Interpretation of in vitro intrinsic clearance between the strains for each species was consistent. Using strict classes of in vitro hepatic intrinsic clearance values (<50, 50-100, >150µL/min/mg protein) comparisons across different conditions such as, assay variables, human lots, mouse and rat strains showed >80% agreement. DISCUSSION: A high throughput assay was developed that enables the simultaneous measurement of CLint using mouse, rat and human hepatic microsomes (consolidated assay). By condensing all possible variables into one assay format, consistent data were obtained during head to head tests.

New Psychoactive Substances 3-Methoxyphencyclidine (3-MeO-PCP) and 3-Methoxyrolicyclidine (3-MeO-PCPy): Metabolic Fate Elucidated with Rat Urine and Human Liver Preparations and their Detectability in Urine by GC-MS, "LC-(High Resolution)-MSn" and "LC-(High Resolution)-MS/MS".

BACKGROUND: 3-Methoxyphencyclidine (3-MeO-PCP) and 3-methoxyrolicyclidine (3-MeOPCPy) are two new psychoactive substances (NPS). The aims of the present study were the elucidation of their metabolic fate in rat and pooled human liver microsomes (pHLM), the identification of the cytochrome P450 (CYP) isoenzymes involved, and the detectability using standard urine screening approaches (SUSA) after intake of common users' doses using gas chromatography-mass spectrometry (GC-MS), liquid chromatography-multi-stage mass spectrometry (LC-MSn), and liquid chromatography-high-resolution tandem mass spectrometry (LC-HR-MS/MS). METHODS: For metabolism studies, rat urine samples were treated by solid phase extraction or simple precipitation with or without previous enzymatic conjugate cleavage. After analyses via LC-HR-MSn, the phase I and II metabolites were identified. RESULTS: Both drugs showed multiple aliphatic hydroxylations at the cyclohexyl ring and the heterocyclic ring, single aromatic hydroxylation, carboxylation after ring opening, O-demethylation, and glucuronidation. The transferability from rat to human was investigated by pHLM incubations, where Odemethylation and hydroxylation were observed. The involvement of the individual CYP enzymes in the initial metabolic steps was investigated after single CYP incubations. For 3-MeO-PCP, CYP 2B6 was responsible for aliphatic hydroxylations and CYP 2C19 and CYP 2D6 for O-demethylation. For 3-MeO-PCPy, aliphatic hydroxylation was again catalyzed by CYP 2B6 and O-demethylation by CYP 2C9 and CYP 2D6 Conclusions: As only polymorphically expressed enzymes were involved, pharmacogenomic variations might occur, but clinical data are needed to confirm the relevance. The detectability studies showed that the authors' SUSAs were suitable for monitoring the intake of both drugs using the identified metabolites.

Defensive nature of Sargassum polycystum (Brown alga) against acetaminophen-induced toxic hepatitis in rats: role of drug metabolizing microsomal enzyme system, tumor necrosis factor-alpha and fate of liver cell structural integrity.

AIM: To assess the defensive nature of Sargassum polycystum (S. polycystum) (Brown alga) against acetaminophen (AAP)-induced changes in drug metabolizing microsomal enzyme system, tumor necrosis factor (TNF-alpha) and fine structural features of the liver during toxic hepatitis in rats. METHODS: Male albino Wistar strain rats used for the study were randomly categorized into 4 groups. Group I consisted of normal control rats fed with standard diet. Group II rats were administered with acetaminophen (800 mg/kg body weight, intraperitoneally). Group III rats were pre-treated with S. polycystum extract alone. Group IV rats were orally pre-treated with S. polycystum extract (200 mg/kg body weight for 21 d) prior to acetaminophen induction (800 mg/kg body weight, intraperitoneally). Serum separated and liver was excised and microsomal fraction was isolated for assaying cytochrome P450, NADPH Cyt P450 reductase and b(5). Serum TNF-alpha was detected using ELISA. Fine structural features of liver were examined by transmission electron microscopy. RESULTS: Rats intoxicated with acetaminophen showed considerable impairment in the activities of drug metabolizing microsomal enzymes, such as cytochrome P450, NADPH Cyt P450 reductase and b(5) when compared with the control rats. The rats intoxicated with acetaminophen also significantly triggered serum TNF-alpha when compared with the control rats. These severe alterations in the drug metabolizing enzymes were appreciably prevented in the rats pretreated with S. polycystum. The rats pretreated with S. polycystum showed considerable inhibition in the elevation of TNF-alpha compared to the rats intoxicated with acetaminophen. The electron microscopic observation showed considerable loss of structural integrity of the endoplasmic reticulum, lipid infiltration and ballooning of mitochondria in the acetaminophen-intoxicated rats, whereas the rats treated with S. polycystum showed considerable protection against acetaminophen-induced alterations in structural integrity. CONCLUSION: These observations suggest that the animals treated with S. polycystum extract may have the ability to protect the drug metabolizing enzyme system and mitochondrial functional status from free radical attack, thereby showing its defense mechanism in protecting hepatic cells from acetaminophen toxic metabolite N-acetyl-para-benzoquinone-imine (NAPQI).

Effect of N-methyl deuteration on metabolism and pharmacokinetics of enzalutamide.

BACKGROUND: The replacement of hydrogen with deuterium invokes a kinetic isotope effect. Thus, this method is an attractive way to slow down the metabolic rate and modulate pharmacokinetics. PURPOSE: Enzalutamide (ENT) acts as a competitive inhibitor of the androgen receptor and has been approved for the treatment of metastatic castration-resistant prostate cancer by the US Food and Drug Administration in 2012. To attenuate the N-demethylation pathway, hydrogen atoms of the N-CH3 moiety were replaced by the relatively stable isotope deuterium, which showed similar pharmacological activities but exhibited favorable pharmacokinetic properties. METHODS: We estimated in vitro and in vivo pharmacokinetic parameters for ENT and its deuterated analog (d3-ENT). For in vitro studies, intrinsic primary isotope effects (K H/K D) were determined by the ratio of intrinsic clearance (CLint) obtained for ENT and d3-ENT. The CLint values were obtained by the substrate depletion method. For in vivo studies, ENT and d3-ENT were orally given to male Sprague Dawley rats separately and simultaneously to assess the disposition and metabolism of them. We also investigated the main metabolic pathway of ENT by comparing the rate of oxidation and hydrolysis in vitro. RESULTS: The in vitro CLint (maximum velocity/Michaelis constant [V max/K m]) of d3-ENT in rat and human liver microsomes were 49.7% and 72.9% lower than those of the non-deuterated compound, corresponding to the K H/K D value of ~2. The maximum observed plasma concentration, C max, and area under the plasma concentration -time curve from time zero to the last measurable sampling time point (AUC0-t) were 35% and 102% higher than those of ENT when orally administered to rats (10 mg/kg). The exposure of the N-demethyl metabolite M2 was eightfold lower, whereas that of the amide hydrolysis metabolite M1 and other minor metabolites was unchanged. The observed hydrolysis rate of M2 was at least ten times higher than that of ENT and d3-ENT in rat plasma. CONCLUSION: ENT was mainly metabolized through the "parent→M2→M1" pathway based on in vitro and in vivo elimination behavior. The observed in vitro deuterium isotope effect translated into increased exposure of the deuterated analog in rats. Once the carbon-hydrogen was replaced with carbon-deuterium (C-D) bonds, the major metabolic pathway was retarded because of the relatively stable C-D bonds. The systemic exposure to d3-ENT can increase in humans, so the dose requirements can be reduced appropriately.

Freeze-fracture analysis of the effects of intermediates of the phosphatidylinositol cycle on fusion of rough endoplasmic reticulum membranes.

While searching for the identity of the effector of the putative GTP-binding protein involved in fusion of rough endoplasmic reticulum (RER) cell-free incubation conditions were found permitting fusion in a GTP-independent manner. Membrane fusion was obtained using medium required to study synthesis of phosphatidylinositol (PI). We now report on the effects of various co-factors and intermediates of the PI cycle on the interaction of rough microsomes. By freeze-fracture, fusion of rough microsomes was defined as the appearance of fracture-planes of membrane larger than those of unincubated membrane. Cytosine triphosphate (CTP, 3 mM) in the presence of 2 mM MnCl2 was most effective in stimulating fusion. Guanosine triphosphate (GTP) at the same concentration, could substitute for CTP to stimulate fusion, ATP, ITP, UTP and guanosine 5'-[gamma-thio]triphosphate (GTP gamma S) could not. When combined together in the same medium CTP potentiated the effect of GTP. Arachidonic acid (20 micrograms/ml) also stimulated fusion in the presence of MnCl2. This led to the appearance of large fracture-planes of membrane with a heterogeneous distribution of intramembranous particles. Other saturated fatty acids at the same concentration did not stimulate fusion. Phosphatidylinositol (PI, 50 micrograms) and 2 mM MnCl2 had a similar effect as arachidonic acid and MnCl2 in stimulating fusion. The PI effect was largely augmented in the presence of CTP. Our results are consistent with the concept that metabolism of phospholipids may modulate GTP-dependent fusion of RER membranes.

Activation of purified 3-hydroxy-3-methylglutaryl-CoA reductase by phospholipids.

3-Hydroxy-3-methylglutaryl-CoA reductase (HMG-CoA reductase), the enzyme that catalyzes the rate-limiting step in cholesterol biosynthesis, has been purified by two previously reported procedures. Enzyme purified by the method of Heller, R. and Shrewsbury, M. (1976) J. Biol. Chem. 251, 3815-3822) shows up to 3-fold enhancement of activity by various types of lipid dispersions while the enzyme purified by the procedure of Tormanen et al. ((1976) Biochem. Biophys. Res. Commun. 68, 754-762) shows no activation. These results suggest that interaction with microsomal membrane lipids may be important in determining the activity of this enzyme. Analysis of bound lipid showed that enzyme prepared by the procedure of Tormanen contained at last 50 times as much phospholipid on a weight basis as enzyme prepared by Heller and Shrewsbury. Analysis of both preparations by gel-electrophoresis indicates that enzyme activities of the two comigrate, but in neither case does activity coincide with the major protein species.

In vitro modification of cholesterol content of rat liver microsomes. Effects upon membrane 'fluidity' and activities of glucose-6-phosphatase and fatty acid desaturation systems.

The cholesterol content of rat liver microsomal membranes was modified in vitro by incubating microsomes and cytosol with liposomes prepared by sonication of microsomal lipids and cholesterol. In this way, the cholesterol to phospholipid molar ratio was increased from 0.11-0.13 in untreated microsomes to a maximal of 0.8 in treated ones. Cholesterol incorporation in microsomes produced an increase in the diphenyl-hexatriene steady-state fluorescence anisotropy and a decrease in the efficiency of pyrene-excimer formation which indicated a decrease in the rotational and translational mobility, respectively, of these probes in the membranes lipid phase. Cholesterol incorporation in microsomes did not affect significantly the glucose-6-phosphatase activity in 0.1% Triton X-100 totally disrupted microsomes, but diminished the glucose-6-phosphatase activity of 'intact' microsomes. This indicates that possibly the glucose 6-phosphate translocation across the microsomal membrane is impeded by an increase in the membrane apparent 'microviscosity'. Cholesterol incorporation in microsomes decreased NADH-cytochrome c reductase without affecting NADH-ferricyanide reductase activity. The delta 9 desaturation reaction rate was enhanced by cholesterol incorporation at low but not at high palmitic acid substrate concentration. delta 5 and delta 6 desaturase reaction-rates were increased both at low and high fatty acid substrate concentrations. These results suggest that a mechanism involving fatty acid desaturase enzymes, might exist to self-regulate the microsomal membrane lipid phase 'fluidity' in the rat liver.

Short-chain aliphatic alcohols increase rat-liver microsomal membrane fluidity and affect the activities of some microsomal membrane-bound enzymes.

n-Butyl and isoamyl alcohols decrease the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene and enhance the efficiency of pyrene excimer formation when these probes are incorporated in rat-liver microsomal membrane, suggesting an increase in rotational and translational mobilities. Neither alcohol modifies NADH-ferricyanide reductase activity but both increase NADH-cytochrome c reductase activity. This was interpreted as an increase in the rate of lateral diffusion of the proteins cytochrome b5 and cytochrome b5 reductase as a consequence of the enhanced membrane lipid phase fluidity. Microsomal delta 9 and delta 6 desaturase activities in the presence of isoamyl alcohol were also studied. This alcohol decreases delta 9 desaturation when it is measured at a low substrate concentration (13 microM palmitic acid), but it is not modified when it is measured at a high substrate concentration (66 microM palmitic acid). delta 6 desaturation is diminished by isoamyl alcohol when it is measured with both 13 microM and 66 microM linoleic acid. The influence of isoamyl alcohol on the glucose-6-phosphatase system activity was also studied. In non-detergent-treated microsomes, isoamyl alcohol enhances glucose-6-phosphatase activity. However, if microsomes are previously treated with 0.1% Triton X-100 isoamyl alcohol does not modify this activity. The enhancement of the glucose 6-phosphate transport rate is not due to membrane permeability barrier disruption, since isoamyl alcohol does not modify mannose-6-phosphohydrolase latency. This would suggest that an increase in membrane lipid phase fluidity specifically activates glucose 6-phosphate transport across the membrane.