Decreased Biosynthesis of Lung Surfactant Constituent Phosphatidylcholine Due to Inhibition of Choline Transporter by Gefitinib in Lung Alveolar Cells

Purpose We investigated whether gefitinib, an anticancer agent, inhibits phosphatidylcholine (PC) biosynthesis and choline uptake by alveolar epithelial type II cells. Materials and Methods Uptake of choline and PC biosynthesis were examined in vitro, using human alveolar epithelia-derived cell line A549 and rat alveolar type (AT) II cells as models. Results Gefitinib reduced the incorporation of [³H]choline into PC in A549 and rat ATII cells. The uptake of [³H]choline by A549 and rat ATII cells was concentration-dependent, and the Km values were 15.0 and 10–100 μM, respectively. The uptake of [³H]choline by A549 and rat ATII cells was weakly Na⁺-dependent, and inhibited by hemicholinium-3. RT-PCR revealed expression of choline transporter-like protein (CTL)1 and organic cation transporter (OCT)3 mRNAs in both cells. The choline uptake by A549 and rat ATII cells was strongly inhibited by gefitinib with the IC₅₀ value of 6.77 μM and 10.5 μM, respectively. Conclusions Our results demonstrate that gefitinib reduces PC biosynthesis via inhibition of cellular choline uptake by A549 and rat ATII cells, which is mainly mediated by CTL1, resulting in abnormality of lung surfactant that can be one of mechanisms of the interstitial lung disease associated with gefitinib.     

Effect of typical inducers of microsomal drug-metabolizing enzymes on phospholipid metabolism in rat liver.

The effect of administration of phenobarbital (PB), polychlorinated biphenyls(PCBs) or 3-methyl-cholanthrene (3-MC) on the metabolism of phospholipids in rat liver was studied by the i.p. injection of [³²P]orthophosphate or [Me-¹⁴C]choline chloride. The inducers were given to animals for 2 successive days PB had no significant effect on the incorporation rate of ³²Pi into liver microsomal phospholipid classes 48 hr after the first administration. The rate of incorporation of [¹⁴C]choline into phosphatidylcholine (PC) in both subcellular components of the liver and blood plasma decreased slightly at this experimental period. In addition, the ratio of [¹⁴C] sp. act. of phosphorylcholinc to that of microsomal phosphulipid was considerably higher on PB-trcated rats as compared with the ratio in control rats. These and previous findings strongly suggest that proliferation of liver endoplasmic reticulum (ER) membranes induced by PB would be accompanied by the stimulation of phospholipid synthesis at the early process of induction and subsequently followed by the decrease in turnover rate of microsomal phospholipids. The administration of PCBs, on the other hand, caused strong inhibition of both ³²Pi and ¹⁴C incorporation into PC in liver subcellular fractions. The secretion of PC from liver cells to blood plasma was also strongly depressed. In addition, phospholipid catabolizing activity was found to be depressed in the liver. These results indicate that hypertrophy of ER membranes in the liver after PCBs administration could be due to a depression of both secretion of lipoprotein from liver cells to plasma and catabolic activity toward membranous phospholipids in liver cells. The decrease in ³²Pi incorporation into liver microsomal PC was also observed in rats treated with 3-MC. There occurred a considerable accumulation of ¹⁴C activity in phosphorylcholine in the liver of rats treated with either PCBs or 3-MC, suggesting strongly that these drugs caused an inhibition of PC synthesis at the site of CDP-choline formation, namely the inhibition of the reaction catalyzed by cholinephosphate cytidylyltransferase.     

Influence of choline,hemicholinium-3 and naphthylvinylpyridine on uptake and acetylation of 3H-labeled choline into hippocampus slices

In slices of rat hippocampus the influence of choline (Ch), hemicholinium-3 (HC-3), and naphthylvinylpyridine (NVP) on the uptake of [³H]-Ch and its incorporation into acetylcholine (ACh) was studied. Increasing concentrations of Ch, added to the incubation medium, led to an elevation of the uptake of labeled precursor and its acetylation. The release of radioactive labeled ACh from slices of hippocampal tissue was also enhanced. A kinetic analysis of the transport of Ch revealed two distinct uptake systems for the precursor with K_m-values of 0.87 μM and 30.3 μM, respectively. The uptake of tritiated Ch into hippocampal slices could be inhibited by HC-3. NVP, an inhibitor of choline acetyltransferase, reduced the uptake of Ch to a similar degree as observed by HC-3, whereas the subsequent acetylation to ACh was not affected. Using an intraventricular application of 400 nmoles unlabeled Ch, the influence of the precursor on uptake and incorporation of [³H]-Ch was also tested in vivo. Both the uptake of Ch and its subsequent acetylation were enhanced, whereas no changes in the ACh content of the hippocampus were observed. The data suggest that the increase of the precursor supply yields an elevation of Ch transport, its acetylation and the transmitter release.     

Action of hemicholinium-3 on phospholipid metabolism in Krebs II ascites cells

Incorporation of [Me-14C]choline or/and [2-14C]ethanolamine into phospholipids of Krebs II ascites cells in toto have been tested in the presence of hemicholinium-3. With [Me-14C]choline, labelling of cell pellet, intracellular choline, phosphocholine and total lipid extract is inhibited by hemicholinium-3 in a dose-dependent way between 6.25 X 10(-6) M and 10(-3) M. These effects are caused by a diminution of the choline or/and ethanolamine transport across the cell membrane and by a choline-kinase inhibition. In Krebs cells, choline is taken up by a low affinity Na+ sensitive uptake system (KT = 46 X 10(-6) M) which is competitively inhibited by hemicholinium-3 (KTi = 161 X 10(-6) M). Krebs cells exert a counter-transport (i.e. an exchange of choline across the membrane) against a concentration gradient of 10 mM choline whereas 10 mM hemicholinium-3 has no effect. Choline-kinase is also inhibited (I50 = 57 X 10(-6) M) in Krebs cells in toto and time-course data suggest that choline transport and phosphorylation might be tightly coupled. Specific radioactivities of phosphocholine and choline-glycerophospholipids decrease owing to the effect of the drug on the uptake and phosphorylation system. With 4 X 10(-5) M hemicholinium-3 and [Me-14C]choline as a marker, labelled choline-glycerophospholipids are decreased by 22%. With [2-14C]ethanolamine, labelled ethanolamine-phospholipids are decreased by 26% and choline-glycerophospholipids remain unlabelled. With the two markers, the additional effect produces a 35% decrease. It is concluded that hemicholinium-3 might be able to induce a depression of the intracellular choline and phosphocholine pool which could provoke a serious quantitative deficiency of major phospholipids in Krebs cells.     

Acetylseco hemicholinium-3, a new choline acetyltransferase inhibitor useful in neuropharmacological studies

Described are the synthesis and some aspects of the pharmacology of acetylseco hemicholinium-3 (acetylseco HC-3), the acetylated open ring analogue of hemicholinium-3 (HC-3). The effects of both compounds were determined in vivo on rat brain acetylcholine (ACh), ¹⁴C-eholine (¹⁴C-Ch) incorporation into ¹⁴C-acetylcholine (¹⁴C-ACh) and on one way jump box avoidance and escape behavior in naive and trained rats. In addition, the in vitro effects of both drugs were determined on choline acetyltransferase activity (ChAc) in rat brain.When given intraventricularly in doses of 1–20 μg both compounds reduced total ACh content in the brain to a maximum of 50% of normal in 30–60 min. In doses of 20 μg intraventricularly, both drugs also reduced ¹⁴C-Ch incorporation into ¹⁴C-ACh by 84.5% for acetylseco HC-3 and by 52% for HC-3.The in vivo changes of ACh in the brain were correlated with the behavioral deficits induced in one way shuttle box acquisition and retention. In doses of 20 μg total intraventricularly, both compounds produced behavioral deficits which were greater in naive than in trained animals. In vitro, acetylseco HC-3 inhibited ChAc activity with an I₅₀ of 1 × 10^?5m with Ch 10^?2m and acetyl CoA 6.4 × 10^?4m, while HC-3 had no inhibitory effects. Using rat brain homogenate as the enzyme source and commercial acetyl CoA for kinetic studies, acetylseco HC-3 was shown to be a mixed inhibitor of acetyl CoA and a competitive inhibitor of Ch.The in vivo actions of acetylseco HC-3 are consistent with those of a ChAc inhibitor. However, it is necessary to rule out the possibility that the drug may also compete with Ch for its transport across biological membranes like its deacetylated derivative HC-3.     

The metabolism of choline in regions of rat brain and the effect of hemicholinium-3.

When [Me-¹⁴C]choline was injected intracerebrally, the radioactivity rapidly spread throughout the brain but the distribution in the six regions examined was not uniform. HC-3 caused a higher retention of the labelled choline particularly in the side of the brain which was the site of the injection. In the cerebellum and mid-brain the synthesis of phosphorylcholine was inhibited by HC-3 but the synthesis of phosphatidylcholine was stimulated. This may be due to an increase in base exchange in the presence of HC-3. In the cortex, phosphorylcholine synthesis was inhibited by HC-3 but no stimulation of phosphatidylcholine synthesis was found. This regional difference may demonstrate that the pathways for the formation of phosphatidylcholine are not the same throughout the brain.     

(3H)Choline entry and (3H)acetylcholine formation in leech segmental ganglia.

Choline entry into the cells of segmental ganglia of the leech, Hirudo medicinalis was studied. Kinetic data obtained for two different choline concentration ranges suggest that there are two different Michaelis constants for the carrier-mediated choline entry, one about 860 μM and the other about 45 μM. The percentage of labelled choline transformed into acetycholine was considerably higher if the ganglia were incubated in 6 μM [³H]choline than in 250 μM [³H]choline. Hemicholinium-3 at 10 and 100 μM inhibited choline entry rate and diminished the percentage of labelled choline transformed into acetycholine. Removal of sodium from the incubating medium affected the choline entry to roughly the same degree at low and at high choline concentrations. The percentage of labelled choline transformed into acetylcholine did not diminish in the sodium-free medium. The results might be interpreted in terms of two choline entry systems in leech segmental ganglia. The choline entry system with the high affinity seems to be linked to acetylcholine synthesis.     

Mechanism of carbachol-stimulated diacylglycerol formation in rat parotid acinar cells

We studied the relationship between phosphoinositide hydrolysis, phosphatidylcholine hydrolysis, and sn-1,2-diacylgglycerol (DAG) formation in response to carbachol stimulation in rat parotid acinar cells. Previously, we demonstrated that DAG formation stimulated with 1 μM carbachol was biphasic: the first peak occurred at 5 min and the second one at 20 min. It was also demonstrated that the second peak was regulated in part by a calmodulin/protein kinase C-dependent mechanism. Based on the kinetic analysis of DAG formation and [³²P]phosphoinositide breakdown, the first peak of carbachol (1 μM)-stimulated DAG accumulation was found to be related to the breakdown of [³²P]phosphatidylinositol 4-monophosphate ([³²P]PIP) and [³²P]phosphatidylinositol 4,5-biphosphate ([³²P]PIP₂). The second peak was found to be related to [³²P]PIP₂ breakdown. Carbachol stimulated the release of [³H]phosphocholine into the medium, indicating that the predominant pathway for phosphatidylcholine hydrolysis was via phospholipase C. Moreover, carbachol stimulated the release of [³H]choline metabolites in a time- and dose-dependent manner. This agonist slightly stimulated the release of [³H]ethanolamine metabolites. A calmodulin/protein kinase C-dependent mechanism was also studied and was found to be involved in carbachol-stimulated phosphatidylcholine hydrolysis; W-7, a calmodulin inhibitor and staurosporine, a protein kinase C inhibitor, inhibited the carbachol (1-μM)-induced release of [³H]choline metabolites at 20 min in a dose-dependent manner, but did not have inhibitory effects at 5 min. These results suggest that the first peak of DAG accumulation induced by carbachol is predominantly associated with the breakdown of [³²P]PIP and [³²P]PIP₂ and that the second peak is predominantly associated with [³²P]PIP₂ breakdown and phosphatidylcholine hydrolysis.     

Factors influencing the microsome- and mitochondria-catalyzed in vitro binding of diethylnitrosamine and N-nitrosopiperidine to deoxyribonucleic acid.

[¹⁴C]Diethylnitrosamine ([¹⁴C]DEN) and [¹⁴C]N-nitrosopiperidine ([¹⁴]NPiP) bind covalently to calf thymus DNA in an in vitro incubation system containing rat liver microsomes. The reaction is NADPH-dependent. Pretreatment of the animals with phenobarbital (PB) enchances the binding of both DEN and NPiP to DNA, whereas the binding of DEN to DNA decreases after 3-methylcholanthrene pretreatment. The PB effect, as observed from the binding of DEN to DNA. is more pronounced in young rats than in the older animals. Addition of cytosol to the incubation system enhances the binding of DEN 3- to 4-fold and the binding of NPiP 2- to 3-fold. Addition of mitochondria to the incubation system increases the binding of [¹⁴C]DEN only slighty. but increases the binding of NPiP more than 5-fold. Addition of mitochondria has no effect on the binding of [¹⁴C]dimethylnitrosamine ([¹⁴C]DMN). Mitochondria alone markedly catalyze the binding of NPiP to DNA. Addition of benzylamine. which is a substrate of mitochondrial monoamine oxidase as well as an inhibitor of DMN-demethylase, inhibits the binding of NPiP catalyzed by microsomes and microsomes plus mitochondria.     

Further studies on the biochemical characterization and autoradiographic distribution of [H]hemicholinium-3 binding sites in rat brain: a presynaptic cholinergic marker

Hemicholinium-3 (HC-3) is a potent inhibitor of the high-affinity choline uptake system (HACU). Here we report on the biochemical characterization and autoradiographic distribution of [3H]hemicholinium-3 binding sites in rat brain, confirming and expanding results from previous studies. The binding of [3H]HC-3 to striatal membranes was specific, to a single site, sodium-dependent, saturable, and of high-affinity, Kd values being about 3 nM for striatum, 5 nM for the hippocampus and 12 nM for neocortex. [3H]HC-3 specific binding exhibited a pharmacological profile suggestive of physiologically relevant interactions and fully comparable to that reported for HACU. The uneven distribution of [3H]HC-3 binding sites exhibited a high degree of correspondence with the reported distribution of HACU and other enzymatic presynpatic cholinergic markers. The punctual differences between our study and previous works on [3H]HC-3 binding are analysed. We conclude that [3H]HC-3 labelling may be used as a selective and quantifiable marker of the cholinergic presynaptic terminals in close relationship with HACU.     

Accumulation and metabolism of choline and homocholine by mouse brain subcellular fractions

Minces of mouse forebrain were incubated in Krebs or a Krebs solution containing high K⁺ (32.7mM) and Li⁺, instead of Na⁺, for 30 min at 37°; subcellular fractions were prepared, and the levels of ACh in the S₃ and P₃ fractions were determined and compared for the two treatments. Incubation of minces in the Krebs solution with high K⁺ and Li⁺, instead of Na⁺, depleted the ACh content of the P₃ fraction 70 per cent, without altering that of the S₃ fraction with respect to incubation of minces in normal Krebs. Subsequent incubation of the depleted minces in normal Krebs containing [¹⁴C]choline (0.1 mM) and paraoxon (0.1μM) refilled the depleted P₃ fraction with newly synthesized [¹⁴C]ACh, and the ratio of [¹⁴C]ACh to total ACh in this fraction (0.63) exceeded that of the S₃ (0.33). Incubation of depleted minces in normal Krebs solution containing the choline analog [¹⁴C]homocholine (0.1 mM) and paraoxon (0.1μM) also refilled the depleted P₃ fraction with newly synthesized [¹⁴C]acetylhomocholine, and the ratio of [¹⁴C]acetylhomocholine to ACh in this fraction (7.26) exceeded that of the S₃ (0.44). Refilling of the depleted P₃ fraction was due to an increase in the accumulation of precursor ([¹⁴C]choline 84 per cent and [¹⁴C]homocholine, 76 per cent) which occurred independently of the S₃. Incubation of depleted minces with either extracellular [¹⁴C]ACh or [¹⁴C]acetylhomocholine did not refill the depleted P₃ fraction with these compounds. These results suggest that ACh, lost from the crude vesicular fraction, can be replaced with newly synthesized ACh independently of the cytoplasm.     

Effect of polychlorinated biphenyls (PCBs) administration on phospholipid biosynthesis in rat liver

The effect of PCBs or phenobarbital on the biosynthesis of phospholipids in hepatic endoplasmic reticulum of rats was studied by the intraperitoneal injection of [³²P]orthophosphate, [Me?¹⁴ C]choline or [2?³H]glycerol. Significant increases in liver microsomal phospholipid content after the administration of either PCBs or phenobarbital indicated the actual proliferation of endoplasmic reticulum membranes. The rate of both [³²P] and [¹⁴C] incorporations into microsomal choline-containing phospholipids, such as phosphatidylcholine, sphingomyelin and lysophosphatidylcholine, was reduced to one fifth by PCBs administration compared with control animals. The incorporation of [³²P]orthophosphate into phosphatidylethanolamine or other phospholipid classes was less or not affected, respectively, by PCBs administration. The specific inhibitory effect of PCBs on the incorporation into cholinecontaining phospholipids was not observed when [2?³-H]glycerol was used as a precursor. Phenobarbital administration, however, increased significantly the rate of [³²P] incorporation into liver phospholipids, especially phosphatidylcholine. It is suggested that the increase in microsomal phospholipid content by PCBs administration is not due to the stimulation of synthesis but to the inhibition of the catabolism of membrane phospholipids and that the increase in content caused by phenobarbital is due at least in part, to the stimulation of synthesis. The possible site(s) of PCBs-induced inhibition of phospholipid biosynthesis in rat liver is discussed.     

Characterization of enzymatic acetylcholine synthesis by mouse brain,rat sperm,and purified carnitine acetyltransferase

Enzymatic acetylcholine synthesis by mouse brain, rat sperm, and purified pigeon breast muscle carnitine acetyltransferase was monitored. Optimal assay procedure, choline substrate requirements, the extent of acetylcholine synthesis, and the effects of acetyltransferase inhibitors were investigated to determine if acetylcholine in rat sperm is synthesized by choline acetyltransferase or by another enzyme that utilizes choline as a substrate. Of two assay procedures tested, the liquid cation-exchange procedure utilizing a butyronitrile-tetraphenylboron extraction was judged superior to an anion-exchange resin procedure. The latter procedure gave falsely high acetylcholine synthesis readings due to another acetylated contaminant (probably acetylcarnitine). The K_m for choline substrate in acetylcholine syntheses by mouse brain, which is a source of choline acetyltransferase, was 0.623 mM choline; this was 300 times less than the choline substrate K_m with rat sperm (207 mM choline) and 80 times less than the K_m with purified carnitine acetyltransferase (50.6 mM choline). Rat sperm had a V_max[3718 pmoles AcCh · min^?1 · (mg protein)^?1] that was > 2-fold that of mouse brain [1603 pmoles AcCh · min^?1·(mg protein)^?1]. A specific inhibitor of choline acetyltransferase, 4-(1-naphthylvinyl)pyridine (500 μM), abolished acetylcholine synthesis by mouse brain, but it caused only a 52 per cent inhibition of acetylcholine synthesis by rat sperm and only a 12 per cent inhibition of acetylcholine synthesis by purified carnitine acetyltransferase. These data indicate that an enzyme other than classical or “true” choline acetyltransferase is responsible for acetylcholine synthesis by rat sperm. Because of the high content of carnitine acetyltransferase in rat sperm and the ability of carnitine acetyltransferase from pigeon breast muscle to synthesize acetylcholine, carnitine acetyltransferase is the most probable enzyme responsible for acetylcholine synthesis in rat sperm.     

Effect of morphine in vivo on uptake of [3H]choline and release of [3H]acetylcholine from rat striatal synaptosomes

The effect of morphine on the rat striatal cholinergic system was investigated in vitro by measuring the rates of [³H]choline uptake and [³H]acetylcholine release in striatal synaptosomes after in vivo injections of morphine sulfate. Morphine caused a 50 per cent increase in the V_max of [³H]choline uptake. Although a concomitant increase was also measured in the amount of [³H] acetylcholine released, it could be explained by the previous increase in uptake. It is suggested that morphine had an overall stimulatory effect on the striatal cholinergic system which may be a transynaptic phenomenon rather than a direct effect on the cholinergic cell.     

Inhibition of choline acetyltransferase by quaternary ammonium analogues of choline.

The effects of the choline analogues hemicholinium-3, triethylcholine, tetraethylammonium and acetylcholine on the synthesis of ACh by choline acetyltransferase have been studied in vitro. Hemicholinium-3 is shown to be an inhibitor of choline acetyltransferase with a K_i of 2·5 mM. HC-3, TEA and ACh are competitive with choline. Michaelis constants for HC-3 and TEC are also calculated and show that these two compounds are competitive substrates. A comparison of the in vitro with the known in vivo behaviour of these compounds is made and the probability stressed that their in vivo inhibitory effect on ACh synthesis is due to inhibition of ChAc itself. The results favour this explanation than the hypothesis that inhibition occurs because of interference with choline transport.     

Some pharmacological actions of acetylsecohemicholinium

The effects of the acetylated derivative of HC-3 (acetylsecohemicholinium; AcHC-3) have been studied at cholinergic nerve terminals and compared with the effects of the parent compound. AcHC-3 blocked neuromuscular transmission in nerve-muscle preparations; it was shown to be less effective than HC-3 in producing a pre-junctional block in the rat diaphragm but was more effective than HC-3 in eliciting a post-junctional blocking effect in the chick biventer muscle. On the frog rectus abdominis muscle AcHC-3 caused a substantial potentiation of the contractures elicited by acetylcholine but did not by itself cause a contracture of the muscle. AcHC-3 inhibited the synthesis of acetylcholine by cholinergic nerve ending particles and inhibited the uptake of [¹⁴C]choline into brain synaptosomal fractions to a similar extent to HC-3. AcHC-3 was shown to be a substrate for cholinesterase enzymes although the rate of hydrolysis was much less than the rate of hydrolysis of acetylcholine. It is concluded that AcHC-3 is effective in inhibiting cholinergic transmission and this acticn is exerted by the open chain (seco) compound and is not due to the hydrolysis of the AcHC-3 by cholinesterases to form the active HC-3 molecule.     

Choline dehydrogenase: Assay,properties and inhibitors

Although choline has proven useful in the treatment of neurological disorders, its duration of action is limited by its rapid rate of metabolism. In order to evaluate the role of choline dehydrogenase in regulation of choline metabolism, some of the properties of the enzyme were studied using a new radioisotopic assay that is more sensitive and specific than previous methods. Choline dehydrogenase activity was present predominantly in kidney and liver of all species examined. Rats, mice, toads, cats, dogs, monkeys and sheep had highest activities in liver, whereas humans, baboons, rabbits and guinea pigs had more enzyme in kidneys. Enzyme activity was found to increase with age, reaching a maximum concentration at 1 week in livers and 4 weeks in kidneys of mice. Choline dehydrogenase, partially purified from rat liver mitochondria, was optimally active at a pH between 7 and 9, at a temperature of 37°, and in the presence of phenazine methosulfate. The K_m for choline was similar for both the soluble (5.7mM) and the particulate (5.3mM) enzyme but the V_max was slightly higher for the soluble [77 nmoles/min · (mg protein)^?1] as compared to the particulate [34 $\text{nmoles}\text{min}$ · (mg protein)^?1] preparation. When choline oxidation was studied in vivo, the concentration of free choline in liver (60 μM) and the rate of oxidation of [³H]-choline administered intravenously [0.29 nmoles/min · (mg protein)^?1] were found to be markedly less than the respective K_m and V_max values determined in vitro. Thus, choline dehydrogenase may not be saturated with choline under physiologic conditions, which could explain the rapid rate of metabolism of exogenously administered choline. The product of the reaction, betaine aldehyde, was a potent competitive inhibitor of choline dehydrogenase.(K_i = 0.6 mM), whereas other products of choline metabolism were weaker inhibitors (K_i values >10 mM). Dimethylaminoethanol (deanol; a cholinomimetic agent) and 2-amino-2-methylpropanol inhibited the enzyme in vitro with inhibitory constants of 1.8 and 1.4mM, respectively, and caused an increase in concentration of choline in kidney and liver when administered to mice, which suggests that choline dehydrogenase is an important enzyme in regulation of the concentration of free choline in tissues under physiologic conditions.     

Studies on the in vivo and in vitro estrogenic activities of methoxychlor and its metabolites. Role of hepatic mono-oxygenase in methoxychlor activation

The injection of rats with methoxychlor stimulated uterine ornithine decarboxylase (ODC) activity and caused an increase in uterine weight 7 hr after injection. The di-demethylated derivative of methoxychlor {[2,2-bis(p-hydroxyphenyl-1,1,1-trichloroethane] (HPTE)} markedly stimulated rat uterine ODC and enlarged uterine wet weight 6 hr after administration. Because we previously demonstrated that HPTE, but not methoxychlor, inhibited the binding of [³H]estradiol-17β ([³H]E₂) to uterine cytosolic estrogen receptor in vitro, we considered the possibility that the estrogenic activity of methoxychlor in vivo was due to biotransformation of methoxychlor. The evolution of formaldehyde occurred when methoxychlor was incubated with rat hepatic microsomes in the presence of NADPH, indicating that methoxychlor was O- demethylated in vitro. The demethylation of methoxychlor was inhibited when methoxychlor was incubated with microsomes in the presence of hexobarbital or 2-diethylaminoethyl diphenyl-propylacetate hydrochloride (SKF-525A), suggesting the involvement of mono-oxygenase. Furthermore, the demethylated products were resolved by thin-layer chromatography (t.l.c.) into three chromatographically distinct components more polar than methoxychlor. One of the products appears to be the di-demethylated derivative of methoxychlor, since it was chromatographically identical to HPTE in three t.l.c. systems. Each of the three components inhibited [³H]E₂ binding to rat uterine cytosol in vitro; however, the metabolite with an R_f equal to that of HPTE demonstrated equal potency to HPTE with respect to suppression of [³H]E₂ binding to uterine cytosol. The possible involvement of mono-oxygenase in biotransformation in methoxychlor into estrogenic metabolites in vivo is discussed.     

Altered lipid metabolism in 2,5-hexanedione-induced testicular atrophy and peripheral neuropathy in the rat

2,5-Hexanedione (HD) induces testicular atrophy and peripheral neuropathy in rats. Since altered lipid metabolism is frequently associated with these disease states, lipid metabolism was investigated in vitro in testes and sciatic nerves of rats fed 1% HD in the drinking water for 6 weeks. Testes from HD-treated rats were 30–60% smaller and weighed threefold less than testes from pair-fed control (PFC) rats. Compared to testes from PFC rats, testes from HD rats exhibited increased incorporation of [¹⁴C]acetate into phospholipids (344%), triacylglycerols (281%), and cholesteryl esters + hydrocarbons (246%) but decreased incorporation into free fatty acids (25%) and sterols + diacylglycerols (65%). The increased incorporation of [¹⁴C]acetate into phospholipids induced by HD reflected an approximate 300% increase into phosphatidyl choline, lysophosphatidyl choline, phosphatidyl serine + phosphatidyl inositol, and phosphatidyl ethanolamine and a disproportionate 800% increase into sphingomyelin. HD rats exhibited clinical signs of peripheral neuropathy, including everted and flat foot placement and hindlimb weakness; similar changes were not observed in PFC rats. In sciatic nerves, the incorporation of [¹⁴C]leucine was decreased into sterols + diacylglycerols (47%), digitoninprecipitable sterols (45%), and cholesteryl esters + hydrocarbons (40%) in HD compared to PFC rats; incorporation of [¹⁴C]leucine into free fatty acids, triacylglycerols, and phospholipids was similar in HD and PFC rats. In contrast to the testis and nerve, lipid metabolism in the liver was similar in HD and PFC rats. The concentrations of 2,5-hexanedione and 2,5-dimethylfuran, respectively, were 0.6 ± 0.3 and 6.5 ± 0.9 μg/g wet weight in the testes and 3.1 ± 0.4 and 3.0 ± 0.4 μg/g wet weight in the livers of HD rats. The data indicate that altered metabolism is associated with HD-induced testicular atrophy and distal axonopathy.     

EFFECT OF BILE DUCT OBSTRUCTION ON HEPATIC UPTAKE OF 1-METHYL-4-PHENYLPYRIDINIUM IN THE RAT

Previous studies have demonstrated that the organic cation 1-methyl-4-phenylpyridinium (MPP⁺) is avidly taken up by rat freshly isolated hepatocytes through at least two distinct transport mechanisms: the type I hepatic transporter of organic cations and P-glycoprotein. In this study, the effects of extrahepatic cholestasis induced by bile duct ligation for 4 days on the uptake of [³H]MPP⁺by rat freshly isolated hepatocytes and liver slices were determined. Bile duct ligation produced no significant alterations in the characteristics of [³H]MPP⁺uptake by freshly isolated hepatocytes. The strong correlation found between the effect of various drugs on [³H]MPP⁺uptake by hepatocytes from control and treated rats (r=0.958;P<0.0001;n=15) suggests that neither the type I hepatic transporter of organic cations nor P-glycoprotein were affected by bile duct ligation. On the contrary, uptake of [³H]MPP⁺by liver slices was markedly changed after bile duct ligation: (1) there was a significant increase (≅40%) in the amount of [³H]MPP⁺taken up by liver slices from bile duct-ligated rats; (2) there was no correlation between the effect of various drugs on [³H]MPP⁺uptake by liver slices from control and treated rats (r=0.772;P=0.072;n=6). On the basis of (1) the lack of effect of bile duct ligation on [³H]MPP⁺uptake by isolated hepatocytes; and (2) the profound morphological alterations of liver tissue observed 4 days after bile duct ligation (increase in volume density of bile ductules, ductular cells and infiltration of inflammatory cells), we suggest that non-parenchymal liver cells have an important participation in the hepatic uptake of [³H]MPP⁺after bile duct ligation in the rat.