Genetic and biochemical investigation on chloral hydrate in vitro and in vivo

Chloral hydrate (CH), a metabolite of trichloroethylene (TCE), was studied in vitro using the D7 diploid strain of Saccharomyces cerevisiae, with and without a mammalian microsomal activation system (S9 fraction), and in vivo by intrasanguineous host-mediated assay (HMA). The in vivo effects on the hepatic microsomal monooxygenase induced by CH in mice pretreated with beta-naphthoflavone (beta-NF) and Naphenobarbital (PB) were also investigated. Chloral hydrate induced a significant increase of mitotic gene conversion in D7 strain both in vivo and in vitro. The enzymatic determinations in mice showed a decrease in aminopyrine N-demethylase (APD) and p-nitroanisole O-demethylase (p-NAD) activities (about 37% and 29% respectively) after one acute dose of CH. Moreover, stability experiments, carried out in the conditions of the liver microsomal assay (LMA), showed an increase of residual activity, after 1 h of preincubation with respect to the control (about 22% and 9% for APD and p-NAD respectively).     

Ex vivo lipopolysaccharide-induced interleukin-1 secretion from murine peritoneal macrophages inhibited by probucol, a hypocholesterolemic agent with antioxidant properties

Probucol, 4,4'-(isopropylidenedithio)bis(2,6-di-tert-butyl-phenol), has been shown to inhibit atherogenesis in genetically hypercholesterolemic (Watanabe) rabbits. Since atherosclerotic lesions contain macrophages capable of screting interleukin 1 (IL 1) and other cytokines that could contribute to the pathogenesis of the disease, we have investigated whether probucol affects IL 1 secretion. Resident peritoneal macrophages from mice dosed with probucol secreted 40-80% less IL 1 than macrophages from control animals when stimulated in vitro with lipopolysaccharide (LPS). The inhibitory effect of probucol was observed when IL 1 was assayed by the standard bioassay, the thymocyte proliferation assay, or a competitive IL 1 receptor binding assay. Probucol treatment had no effect on LPS-induced membrane IL 1 expression; secretion of tumor necrosis factor (TNF); Con A-induced splenic interleukin 2 (IL 2) and interleukin 3 (IL 3) release; and prostaglandin- or zymosan-induced secretion of prostacyclin, leukotriene C4, acid phosphatase, or superoxide anion. In contrast to the effect of oral administration, direct addition of probucol to macrophage cultures did not inhibit IL 1 release. Probucol administration did, however, inhibit the fall in serum zinc level induced by intravenous injection of LPS in zymosan-primed mice but had no effect on the LPS-induced increase in serum triglyceride levels, which indirectly confirms that probucol administration inhibits IL 1 but not TNF secretion. Paw granuloma induced in mice by heat-killed mycobacteria was inhibited by oral administration of probucol, an effect that may be attributable to inhibition of IL 1 secretion. Probucol neither reduced zymosan-induced liver granulomata in mice nor inhibited adjuvant-induced arthritis in rats. We suggest that inhibition of IL 1 secretion from macrophages by probucol contributes to its therapeutic effects in atherosclerosis and may also result in beneficial activity in some chronic inflammatory diseases.     

Ethanolamine plasmalogens prevent the oxidation of cholesterol by reducing the oxidizability of cholesterol in phospholipid bilayers

The aims of the present study are to establish an appropriate system for assessing the oxidizability of cholesterol (CH) in phospholipid (PL) bilayers, and to explore the effect of ethanolamine plasmalogens on the oxidizability of CH with the system, through comparing with those of choline plasmalogens, phosphatidylethanolamine, and antioxidant alpha-tocopherol (Toc). Investigation of the effects of oxidants, vesicle lamellar forms, saturation level, and constituent ratio of PLs in vesicles on CH oxidation revealed the suitability of a system comprising unilamellar vesicles and the water-soluble radical initiator 2,2'-azobis (2-amidino-propane) dihydrochloride (AAPH). As CH oxidation in the system was found to follow the rate law for autoxidation without significant interference from oxidizable PLs, the oxidizability of CH in PL bilayers could be experimentally determined from the equation: k (p)/(2k (t))(1/2)=R (p)/[LH]R(i)(1/2) by measuring the rate of CH oxidation. It was found with this system that bovine brain ethanolamine plasmalogen (BBEP), bovine heart choline plasmalogen, and egg yolk phosphatidylethanolamine lower the oxidizability of CH in bilayers. Comparison of the dose-dependent effects of each PL demonstrated the greatest ability of BBEP to reduce the oxidizability. A time course study of CH oxidation suggested a novel mechanism of BBEP for lowering the oxidizability of CH besides the action of scavenging radicals.     

Add-On Effect of Probucol in Atherosclerotic,Cholesterol-Fed Rabbits Treated with Atorvastatin

Objective

Lowering the blood concentration of low-density lipoprotein (LDL) cholesterol is the primary strategy employed in treating atherosclerotic disorders; however, most commonly prescribed statins prevent cardiovascular events in just 30% to 40% of treated patients. Therefore, additional treatment is required for patients in whom statins have been ineffective. In this study of atherosclerosis in rabbits, we examined the effect of probucol, a lipid-lowering drug with potent antioxidative effects, added to treatment with atorvastatin.

Methods and Results

Atherosclerosis was induced by feeding rabbits chow containing 0.5% cholesterol for 8 weeks. Probucol 0.1%, atorvastatin 0.001%, and atorvastatin 0.003% were administered solely or in combination for 6 weeks, beginning 2 weeks after the start of atherosclerosis induction. Atorvastatin decreased the plasma concentration of non-high-density lipoprotein cholesterol (non-HDLC) dose-dependently; atorvastatin 0.003% decreased the plasma concentration of non-HDLC by 25% and the area of atherosclerotic lesions by 21%. Probucol decreased the plasma concentration of non-HDLC to the same extent as atorvastatin (i.e., by 22%) and the area of atherosclerotic lesions by 41%. Probucol with 0.003% atorvastatin decreased the plasma concentration of non-HDLC by 38% and the area of atherosclerotic lesions by 61%. Co-administration of probucol with atorvastatin did not affect the antioxidative effects of probucol, which were not evident on treatment with atorvastatin alone, such as prevention of in vitro LDL-oxidation, increase in paraoxonase-1 activity of HDL, and decreases in plasma and plaque levels of oxidized-LDL in vivo.

Conclusions

Probucol has significant add-on anti-atherosclerotic effects when combined with atorvastatin treatment; suggesting that this combination might be beneficial for treatment of atherosclerosis.     

Probucol-Induced α-Tocopherol Deficiency Protects Mice against Malaria Infection

The emergence of malaria pathogens having resistance against antimalarials implies the necessity for the development of new drugs. Recently, we have demonstrated a resistance against malaria infection of α-tocopherol transfer protein knockout mice showing undetectable plasma levels of α-tocopherol, a lipid-soluble antioxidant. However, dietary restriction induced α-tocopherol deficiency is difficult to be applied as a clinical antimalarial therapy. Here, we report on a new strategy to potentially treat malaria by using probucol, a drug that can reduce the plasma α-tocopherol concentration. Probucol pre-treatment for 2 weeks and treatment throughout the infection rescued from death of mice infected with Plasmodium yoelii XL-17 or P. berghei ANKA. In addition, survival was extended when the treatment started immediately after parasite inoculation. The ratio of lipid peroxidation products to parent lipids increased in plasma after 2 weeks treatment of probucol. This indicates that the protective effect of probucol might be mediated by the oxidative stressful environment induced by α-tocopherol deficiency. Probucol in combination with dihydroartemisin suppressed the proliferation of P. yoelii XL-17. These results indicated that probucol might be a candidate for a drug against malaria infection by inducing α-tocopherol deficiency without dietary α-tocopherol restriction.     

Probucol as a potent inhibitor of oxygen radical-induced lipid peroxidation and DNA damage: in vitro studies

Probucol, a clinically used cholesterol lowering and antioxidant drug, was investigated for possible protection against lipid peroxidation and DNA damage induced by iron nitrilotriacetate (Fe-NTA) plus hydrogen peroxide (H2O2). Fe-NTA is a potent nephrotoxic agent and induces acute and subacute renal proximal tubular necrosis by catalyzing the decomposition of H2O2-derived production of hydroxyl radicals, which are known to cause lipid peroxidation and DNA damage. Fe-NTA is associated with a high incidence of renal adenocarcinoma in rodents. Lipid peroxidation and DNA damage are the principal manifestation of Fe-NTA induced toxicity, which could be mitigated by probucol. Incubation of renal microsomal membrane and/or calf thymus DNA with H2O2 (40 mM) in the presence of Fe-NTA (0.1 mM) induces renal microsomal lipid peroxidation and DNA damage to about 2.4-fold and 5.9-fold, respectively, as compared to control (P < 0.05). Induction of renal microsomal lipid peroxidation and DNA damage was inhibited by probucol in a concentration-dependent manner. In lipid peroxidation protection studies, probucol treatment showed a concentration-dependent inhibition (10-34% inhibition; P < 0.05) of Fe-NTA plus H2O2-induced lipid peroxidation as measured by thiobarbituric acid reacting species' (TBARS) formation in renal microsomes. Similarly, in DNA damage protection studies, probucol treatment also showed a concentration-dependent strong inhibition (36-71% inhibition; P < 0.05) of DNA damage. From these studies, it was concluded that probucol inhibits peroxidation of microsomal membrane lipids and DNA damage induced by Fe-NTA plus H2O2. However, because the lipid peroxidation and DNA damage studied here are regarded as early markers of carcinogenesis, we suggest that probucol may be developed as a cancer chemopreventive agent against renal carcinogenesis and other adverse effects of Fe-NTA exposure in experimental animals, in addition to being a cholesterol-lowering drug, useful for the control of hypercholestrolemia.     

Transmembrane topography of plasma membrane constituents in mung bean (Vigna radiata L.) hypocotyl cells. I. Transmembrane distribution of phospholipids.

The transmembrane distribution of phospholipids (PLs) in the plasma membrane (PM) of mung bean (Vigna radiata L.) hypocotyl cells was investigated using annexin V-fluorescein isothiocyanate, porcine pancreas phospholipase A(2), and (31)P-nuclear magnetic resonance (NMR) spectroscopy. Phosphatidylserine was not located on the cell surface of mung bean protoplasts. However, phosphatidylcholine, phosphatidylethanolamine and phosphatidic acid were found to be almost symmetrically distributed across right-side-out PM vesicles obtained by aqueous two-phase partitioning by porcine pancreas phospholipase A(2) assay. (31)P-NMR assay showed that the amount of PLs is about equal in the outer and the inner leaflets of the right-side-out PM vesicles. These results suggest that the topography of PM PLs might not contribute to well-known asymmetrical properties of the outer and inner surfaces of higher plant PMs. It is also indicated that inside-out PM vesicles created by Brij 58-treatment do not retain the native PL topography on dithionate reduction of 7-nitro-2,1,3-benzoxadiazol-4-yl-labeled PLs incorporated in the PM vesicles.     

Permeability changes induced by polylysines in rat spermatids

High molecular weight (HMW, >15 kDa) but not low molecular weight (LMW, <15 kDa) polylysines (PLs) bound and induced permeability changes in rat spermatid plasma membranes, estimated by Mn2+ quenching of intracellular indo-1 fluorescence (K(1/2) = 3.3 +/- 0.5 microg/ml) and Co2+ quenching of intracellular calcein. The pharmacology of the Mn2+ entry pathway activated by HMW PL does not suggest that Ca2+ channels are involved in this phenomenon. Concentrations of HMW PL that induced divalent ion entry did not induce the entry of ethidium bromide, suggesting that HMW PL first bound and perturbed the plasma membrane structure inducing a non-specific increase in membrane permeability. High concentrations of HMW PL induced cell lysis (K(1/2) = 23 microg/ml). The binding of HMW PL, initially homogenous on the cell surface, subsequently progressed to a segregated pattern resembling a clustering phenomenon.     

Two-dimensional electrophoresis of membrane proteins. Factors affecting resolution of rat-liver microsomal proteins

A comparison has been made of published techniques for the resolution of rat liver microsomal proteins by two-dimensional electrophoresis. The method of Kaderbhai and Freedman (Biochim. Biophys. Acta 601 (1980) 21-20) gives good resolution of acidic proteins but excludes hydrophobic integral membrane proteins of pI greater than 7, including cytochrome P-450 apoproteins. The method of Vlasuk and Walz (Anal. Biochem. 105 (1980) 112-120) gives good resolution of proteins of pI 5-8, including cytochromes P-450, but fails to resolve a major acidic protein of pI less than 5. Isoelectric focusing of microsomal proteins is improved by the use of high concentrations of urea and low concentrations of sample proteins. Zwitterionic detergents of the general formula R . N+(CH3)2 . CH2CH2CH2SO3- are effective in solubilizing microsomal proteins, either alone or in presence of non-ionic detergent; compounds with a long alkyl chain (C14 or C16) are most effective. Isoelectric focusing of microsomal proteins solubilized by zwitterionic detergents did not give good resolution, probably because of incomplete dissociation and denaturation of the proteins. These detergents could not be used in the presence of high concentrations of urea. Although no single method of two-dimensional electrophoresis gives complete resolution of the whole range of microsomal proteins, conditions can be optimized for specific sets of proteins of interest. The technique can be used to monitor differences in microsomal composition between rat strains, or following induction, and for a variety of other studies.     

Effects of probucol on atherosclerosis of apoE-deficient or LDL receptor-deficient mice.

The effect of probucol on atheroma formation was evaluated using mouse models for atherosclerosis with different diet protocols. Dietary administration of probucol (0.5 %, wt/wt) for 12 weeks reduced total plasma cholesterol levels in both apolipoprotein E (apoE)-deficient mice fed a western diet and in low-density lipoprotein receptor (LDLR)-deficient mice fed a Paigen diet by 60 % and 30 % to 60 %, respectively. Probucol treatment also significantly reduced high-density lipoprotein (HDL) levels in apoE-deficient mice, but not in LDLR-deficient mice. Atherosclerotic plaques in the aortic sinus of probucol-treated apoE-deficient mice were two-fold larger than those in untreated apoE-deficient mice, while the lesions in probucol-treated LDLR-deficient mice were similar to those in untreated LDLR-deficient mice. A strong negative correlation between HDL cholesterol levels and lesion sizes at the aortic sinus was observed in apoE-deficient mice, but not in LDLR-deficient mice. Thus, in contrast to LDLR-deficient mice, probucol had a strong proatherogenic effect in the aortic sinus of apoE-deficient mice associated with the reduction of HDL levels in spite of the reduction of total plasma cholesterol levels. The varying effects of probucol on atherogenesis depend upon the portion of aorta and which animal model is evaluated, implicating that complex cellular events are involved in the effect of probucol.     

Reduction of hexavalent chromium by human cytochrome b5: generation of hydroxyl radical and superoxide

The reduction of hexavalent chromium, Cr(VI), can generate reactive Cr intermediates and various types of oxidative stress. The potential role of human microsomal enzymes in free radical generation was examined using reconstituted proteoliposomes (PLs) containing purified cytochrome b(5) and NADPH:P450 reductase. Under aerobic conditions, the PLs reduced Cr(VI) to Cr(V) which was confirmed by ESR using isotopically pure (53)Cr(VI). When 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) was included as a spin trap, a very prominent signal for the hydroxyl radical (HO()) adduct was observed as well as a smaller signal for the superoxide (O(2)(-)) adduct. These adducts were observed even at very low Cr(VI) concentrations (10 muM). NADPH, Cr(VI), O(2), and the PLs were all required for significant HO() generation. Superoxide dismutase eliminated the O(2)(-) adduct and resulted in a 30% increase in the HO() adduct. Catalase largely diminished the HO() adduct signal, indicating its dependence on H(2)O(2). Some sources of catalase were found to have Cr(VI)-reducing contaminants which could confound results, but a source of catalase free of these contaminants was used for these studies. Exogenous H(2)O(2) was not needed, indicating that it was generated by the PLs. Adding exogenous H(2)O(2), however, did increase the amount of DEPMPO/HO() adduct. The inclusion of formate yielded the carbon dioxide radical adduct of DEPMPO, and experiments with dimethyl sulfoxide (DMSO) plus the spin trap alpha-phenyl-N-tert-butylnitrone (PBN) yielded the methoxy and methyl radical adducts of PBN, confirming the generation of HO(). Quantification of the various species over time was consistent with a stoichiometric excess of HO() relative to the net amount of Cr(VI) reduced. This also represents the first demonstration of a role for cytochrome b(5) in the generation of HO(). Overall, the simultaneous generation of Cr(V) and H(2)O(2) by the PLs and the resulting generation of HO() at low Cr(VI) concentrations could have important implications for Cr(VI) toxicity.     

Liver-specific deletion of the NADPH-cytochrome P450 reductase gene: impact on plasma cholesterol homeostasis and the function and regulation of microsomal cytochrome P450 and heme oxygenase

A mouse model with liver-specific deletion of the NADPH-cytochrome P450 reductase (Cpr) gene (designated Alb-Cre/Cprlox mice) was generated and characterized in this study. Hepatic microsomal CPR expression was significantly reduced at 3 weeks and was barely detectable at 2 months of age in the Alb-Cre+/-/Cprlox+/+ (homozygous) mice, with corresponding decreases in liver microsomal cytochrome P450 (CYP) and heme oxygenase (HO) activities, in pentobarbital clearance, and in total plasma cholesterol level. Nevertheless, the homozygous mice are fertile and are normal in gross appearance and growth rate. However, at 2 months, although not at 3 weeks, the homozygotes had significant increases in liver weight, accompanied by hepatic lipidosis and other pathologic changes. Intriguingly, total microsomal CYP content was increased in the homozygotes about 2-fold at 3 weeks and about 3-fold at 2 months of age; at 2 months, there were varying degrees of induction in protein (1-5-fold) and mRNA expression (0-67-fold) for all CYPs examined. There was also an induction of HO-1 protein (nearly 9-fold) but no induction of HO-2. These data indicate the absence of significant alternative redox partners for liver microsomal CYP and HO, provide in vivo evidence for the significance of hepatic CPR-dependent enzymes in cholesterol homeostasis and systemic drug clearance, and reveal novel regulatory pathways of CYP expression associated with altered cellular homeostasis. The Alb-Cre/Cprlox mouse represents a unique model for studying the in vivo function of hepatic HO and microsomal CYP-dependent pathways in the biotransformation of endogenous and xenobiotic compounds.     

Inhibition of Lipid Peroxidation by Dihydroquinoline-Type Antioxidant (CH 402)

The in vitro effect of a non-toxic, water soluble, low molecular weight, stable dihydroquinoline-type antioxidant, CH 402 (Na (2,2-dimethyl-1,2-dihydroquinoline-4-yl) –- methane sulphonic acid) was studied on free radical reactions in brain subcellular fractions. Experiments were performed using rat and mouse brain homogenate and microsomal fractions. Non … enzymatically induced lipid peroxidation by ascorbic acid was studied in correlation with ascorbic acid and CH 402 concentrations and incubation time. Malondialdehyde production during lipid peroxidation was measured by the thiobarbituric acid test. In a concentration range of 10^?2–10^?5 M CH 402 dose - dependently inhibited the ascorbic acid induced in vitro lipid peroxidation in mouse and rat brain subcellular fractions.     

The antagonistic effects of 5-ethyl-5-(3-hydroxy-1-methylbutyl)-barbituric acid on pentobarbital narcosis in both naïve and tolerant mice

These studies demonstrated that 5-ethyl-5-(3-hydroxyl-1-methyl-butyl)-barbituric acid (PB-OH), a major metabolite of pentobarbital, antagonized pentobarbital-induced narcosis in both naive and pentobarbital tolerant mice. In PB-OH pretreated mice, the sleeping time induced by sodium pentobarbital was significantly shorter than that of the saline control animals. However, PB-OH failed to modify the pentobarbital-induced hypothermia. The findings also demonstrated that hepatic microsomal enzyme activity and half lives of pentobarbital and PB-OH in both plasma and brain were not modified by the pretreatment of PB-OH. The specific antagonistic effect of PB-OH appears to be a direct effect on sites in the CNS.     

The effect of CH 402 dihydroquinoline type antioxidant on the activity of Na+-K+-ATP-ase and Mg++-ATP-ase of rat brain subcellular fractions in the presence and absence of ascorbic acid

Authors studied the activities of Na+-K+-ATP-ase and Mg++-ATP-ase as indicators of lipid peroxidation on rat brain plasmamembrane and microsomal fraction. The CH 402 (Sodium(2,2-dimethyl-1,2-dihydroquinoline-4-yl)methane sulfonate) a synthetic, water soluble, non toxic dihydroquinoline type antioxidant proved to be effective in decreasing the membrane damage caused by ascorbic acid induced lipid peroxidation. The CH 402 did not inhibit the Na+-K+-ATP-ase and Mg++-ATP-ase activities even at a concentration of 10(-3) mol/l.     

Manipulation of light and CO2 environments of the primary leaves of bean (Phaseolus vulgaris L.) affects photosynthesis in both the primary and the first trifoliate leaves: involvement of systemic regulation.

Possible involvement of systemic regulation of the photosynthetic properties of young leaves by the local environments and/or photosynthate production of the mature leaves were examined using Phaseolus vulgaris plants. When primary leaves (PLs) were treated with air containing 150 microL CO2 L(-1) with the other plant parts in ambient air at a photosynthetic photon flux density (PPFD) of 300 micromol photon m(-2) s(-1), decreases in the photosynthetic rate measured at 360 microL CO2 L(-1) and a PPFD of 300 micromol photon m(-2) s(-1) (A360) were markedly retarded in both PLs and the first trifoliate leaves (TLs) as compared to plants treated with 400 microL CO2 L(-1). Conversely, when PLs were treated with 1000 microL CO2 L(-1), decreases in A360 were accelerated in both PLs and TLs. Shading of PLs accelerated the decrease in PL A360, and delayed the decrease in TLs. In the CO2 treatments, changes in A360 in TLs were mainly attributed to the changes in ribulose bisphosphate (RuBP) carboxylation rate, while the shading of PLs caused increases in both the RuBP carboxylation and regeneration rates in TLs. The ribulose 1.5-bisphosphate carboxylase/oxygenase (Rubisco) activity on chlorophyll basis, an indicator of sun/shade acclimation, differed both among PLs and among TLs in accordance with the redox state of photosystem II (PSII) in PLs. Although carbohydrate contents of TLs were not affected by any manipulation of PLs, changes in the photosynthetic capacities of TLs acted to compensate for changes in PL photosynthesis. These results clearly indicate that the CO2 and shade treatments of PLs not only affect photosynthetic properties of the PLs themselves, but also systemically affected the photosynthetic properties of TLs. Possible roles of the redox state and photosynthate concentration in PLs in regulation of photosynthesis in PLs and TLs are discussed.     

Arylamidases of rat liver and chemically induced hepatomas 1. Subcellular distribution of l-leucine 2. Naphthylamidase-active antigens

The subcellular distribution of arylamidase-active antigens in rat liver and in two chemically induced hepatomas (D23 and D33) was investigated. Soluble antigens or detergent-solubilized membrane antigens from isolated subcellular fractions were tested in fused rocket immunoelectrophoresis against antisera prepared against each of the fractions. The arylamidase active antigens were identified by means of a zymogram technique using l-leucine 2-naphthylamide as substrate.Two arylamidase-active antigens were shown to be shared between plasma membranes, microsomes, lysosomal membranes and lysosomal content of the hepatocytes. One of these occurred predominantly in the plasma membranes (the plasma membrane arylamidase) while the other was preferentially found in the lysosomal content (the lysosomal content arylamidase). Also a third arylamidase-active antigen was identified and was shown to be restricted to the microsomes and the lysosomal membranes (the microsomal/lysosomal arylamidase).The rat liver plasma membrane arylamidase-active antigen was also present in plasma membrane, microsomal an cell-sap fractions of both the hepatomas. However, in the hepatomas this antigen occurred predominantly in the microsomal fraction. The plasma membrane arylamidase was the only arylamidase-active antigen found in the hepatoma D33 while the plasma membrane and microsomal fractions of hepatoma D23 also contained another antigen with this activity. Neither the lysosomal content arylamidase nor the microsomal/lysosomal arylamidase could be detected in any of the hepatoma fractions.     

mPGES-1 deletion potentiates urine concentrating capability after water deprivation

PGE(2) plays an important role in the regulation of fluid metabolism chiefly via antagonizing vasopressin-induced osmotic permeability in the distal nephron, but its enzymatic sources remain uncertain. The present study was undertaken to investigate the potential role of microsomal PGE synthase (mPGES)-1 in the regulation of urine concentrating ability after water deprivation (WD). Following 24-h WD, wild-type (WT) mice exhibited a significant reduction in urine volume, accompanied by a significant elevation in urine osmolality compared with control groups. In contrast, in response to WD, mPGES-1 knockout (KO) mice had much less urine volume and higher urine osmolality. Analysis of plasma volume by measurement of hematocrit and by using a nanoparticle-based method consistently demonstrated that dehydrated WT mice were volume depleted, which was significantly improved in the KO mice. WD induced a twofold increase in urinary PGE(2) output in WT mice, which was completely blocked by mPGES-1 deletion. At baseline, the KO mice had a 20% increase in V(2) receptor mRNA expression in the renal medulla but not the cortex compared with WT controls; the expression was unaffected by WD irrespective of the genotype. In response to WD, renal medullary aquaporin-2 (AQP2) mRNA exhibited a 60% increase in WT mice, and this increase was greater in the KO mice. Immunoblotting demonstrated increased renal medullary AQP2 protein abundance in both genotypes following WD, with a greater increase in the KO mice. Similar results were obtained by using immunohistochemistry. Paradoxically, plasma AVP response to WD seen in WT mice was absent in the KO mice. Taken together, these results suggest that mPGES-1-derived PGE(2) reduces urine concentrating ability through suppression of renal medullary expression of V(2) receptors and AQP2 but may enhance it by mediating the central AVP response.     

Arylamidases of rat liver and chemically induced hepatomas. 1. Subcellular distribution of L-leucine. 2. Naphthylamidase-active antigens.

The subcellular distribution of arylamidase-active antigens in rat liver and in two chemically induced hepatomas (D23 and D33) was investigated. Soluble antigens or detergent-solubilized membrane antigens from isolated subcellular fractions were tested in fused rocket immunoelectrophoresis against antisera prepared against each of the fractions. The arylamidase active antigens were identified by means of a zymogram technique using L-leucine 2-naphthylamide as substrate. Two arylamidase-active antigens were shown to be shared between plasma membranes, microsomes, lysosomal membranes and lysosomal content of the hepatocytes. One of these occurred predominantly in the plasma membranes (the plasma membrane arylamidase) while the other was preferentially found in the lysosomal content (the lysosomal content arylamidase). Also a third arylamidase-active antigen was identified and was shown to be restricted to the microsomes and the lysosomal membranes (the microsomal/lysosomal arylamidase). The rat liver plasma membrane arylamidase-active antigen was also present in plasma membrane, microsomal and cell-sap fractions of both the hepatomas. However, in the hepatomas this antigen occurred predominantly in the microsomal fraction. The plasma membrane arylamidase was the only arylamidase-active antigen found in the hepatoma D33 while the plasma membrane and microsomal fractions of hepatoma D23 also contained another antigen with this activity. Neither the lysosomal content arylamidase nor the microsomal/lysosomal arylamidase could be detected in any of the hepatoma fractions.     

DNA adduct formation by alachlor metabolites

The extent of DNA adduct formation by alachlor [ArN(CH2OCH3)C(O)CH2Cl wherein Ar is 2,6-diethylphenyl] and its metabolites is used as a guide to deduce the causal agent(s) in the carcinogenicity of this major herbicide. [14C-phenyl]Alachlor is compared to its two metabolic cleavage products, [14C-phenyl]2-chloro-N-(2,6-diethylphenyl)acetamide (CDEPA) [ArNHC(O)CH2Cl] and [14C-phenyl]2,6-diethylaniline (DEA) (ArNH2), and to [14C-methoxy]alachlor in various in vitro and in vivo systems. Horseradish peroxidase and hydrogen peroxide activate DEA, but not CDEPA or alachlor, for formation of adducts with calf thymus DNA, which probably involves 2,6-diethylnitrosobenzene (ArNO) as an intermediate. Mouse liver microsomes and NADPH are both required to enhance the binding from each labeled preparation to calf thymus DNA; 4-fold higher labeling is observed from [14C-methoxy]- than from [14C-phenyl]alachlor. This 4-fold preferential DNA labeling from the 14C-methoxy compound is likewise found in the liver of mice treated intraperitoneally. Mouse liver protein and hemoglobin are also labeled, in vivo, with [14C-phenyl]alachlor, -CDEPA and -DEA, and, as with the DNA, the labeling of these proteins is 1.5- to 2-fold higher with [14C-methoxy]alachlor. Metabolic studies indicate that ArN(CH2OCH2OH)C(O)CH2Cl is an intermediate in forming CDEPA and presumably formaldehyde in the mouse liver microsomal mixed-function oxidase system and in yielding the O-glucuronide of ArN(CH2OH)C(O)CH2Cl in the urine of alachlor-treated mice. These findings point to the N-CH2OCH2OH metabolite or formaldehyde as a reactive intermediate in forming a DNA-adduct and as a candidate proximate carcinogen.