Fraction Of Rat Liver Homogenate Research Articles

Riboflavin compounds show NAD(P)H dependent quinone oxidoreductase-like quinone reducing activity.

NAD(P)H-dependent quinone oxidoreductase (NQO) is an essential enzyme in living organisms and cells protecting them from oxidative stress. NQO reduces coenzyme Q (CoQ) using NAD(P)H as an electron donor. In the present study, we searched for coenzyme Q10 reducing activity from fractions of gel filtration-fractionated rat liver homogenate. In addition to the large-molecular-weight fraction containing NQO, CoQ10 reducing activity was also detected in a low-molecular-weight fraction. Furthermore, dicumarol, a conventional inhibitor of NQO1 (DT diaphorase), did not inhibit the reduction but quercetin did, suggesting that the activity was not due to NQO1. After further purification, the NADH-dependent CoQ10-reducing compound was identified as riboflavin. Riboflavin is an active substituent of other flavin compounds such as FAD and FMN. These flavin compounds also reduced not only CoQ homologues but also vitaminK homologues in the presence of NADH. The mechanism was speculated to work as follows: NADH reduces flavin compounds to the corresponding reduced forms, and subsequently, the reduced flavin compounds immediately reduce bio-quinones. Furthermore, the flavin-NADH system reduces CoQ10 bound with saposin B, which is believed to function as a CoQ transfer protein in vivo. This flavin-dependent CoQ10 reduction, therefore, may function in aqueous phases such as the cell cytosol and bodily fluids.

Mechanisms of Fatal Cardiotoxicity following High-Dose Cyclophosphamide Therapy and a Method for Its Prevention

Observed only after administration of high doses, cardiotoxicity is the dose-limiting effect of cyclophosphamide (CY). We investigated the poorly understood cardiotoxic mechanisms of high-dose CY. A rat cardiac myocardial cell line, H9c2, was exposed to CY metabolized by S9 fraction of rat liver homogenate mixed with co-factors (CYS9). Cytotoxicity was then evaluated by 3-(4,5-dimethyl-2-thiazolyl)¬2,5-diphenyl¬2H-tetrazolium bromide (MTT) assay, lactate dehydrogenase release, production of reactive oxygen species (ROS), and incidence of apoptosis. We also investigated how the myocardial cellular effects of CYS9 were modified by acrolein scavenger N-acetylcysteine (NAC), antioxidant isorhamnetin (ISO), and CYP inhibitor β-ionone (BIO). Quantifying CY and CY metabolites by means of liquid chromatography coupled with electrospray tandem mass spectrometry, we assayed culture supernatants of CYS9 with and without candidate cardioprotectant agents. Assay results for MTT showed that treatment with CY (125–500 μM) did not induce cytotoxicity. CYS9, however, exhibited myocardial cytotoxicity when CY concentration was 250 μM or more. After 250 μM of CY was metabolized in S9 mix for 2 h, the concentration of CY was 73.6 ± 8.0 μM, 4-hydroxy-cyclophosphamide (HCY) 17.6 ± 4.3, o-carboxyethyl-phosphoramide (CEPM) 26.6 ± 5.3 μM, and acrolein 26.7 ± 2.5 μM. Inhibition of CYS9-induced cytotoxicity occurred with NAC, ISO, and BIO. When treated with ISO or BIO, metabolism of CY was significantly inhibited. Pre-treatment with NAC, however, did not inhibit the metabolism of CY: compared to control samples, we observed no difference in HCY, a significant increase of CEPM, and a significant decrease of acrolein. Furthermore, NAC pre-treatment did not affect intracellular amounts of ROS produced by CYS9. Since acrolein seems to be heavily implicated in the onset of cardiotoxicity, any competitive metabolic processing of CY that reduces its transformation to acrolein is likely to be an important mechanism for preventing cardiotoxicity.

Mechanisms and Prevention of Fatal Cardiotoxicity Following High-Dose Cyclophosphamide Therapy

Background: High-dose Cyclophosphamide (CY) is a mainstay in most conditioning regimens for hematopoietic stem cell transplantation. Recently, high-dose, posttransplantation CY, a novel effective strategy for preventing graft-versus-host disease, has been attracting a lot of attention. CY is activated by the hepatic cytochrome P-450 (CYP) enzyme system to form 4-hydroxy-cyclophosphamide (HCY), which is in equilibrium with aldocyclophosphamide (AldoCY). Depending on the type of the cell, AldoCY decomposes to form cytotoxic phosphoramide mustard and the byproduct acrolein. Alternatively, aldoCY is oxidized to the inactive metabolite o-carboxyethylphosphoramide mustard (CEPM) by aldehyde dehydrogenase1 (ALDH1). The dose-limiting toxic effect of CY, observed only after administration of high-doses, is cardiotoxicity. Since the mechanism underlying this phenomenon has not yet been elucidated, we investigated the cardiotoxic mechanisms of high-dose CY. To determine preliminary mechanisms that may prevent the occurrence of CY-induced cytotoxicity in H9C2 cells, we also evaluated the protective effects of potential cardioprotectant agents (PCA).Methods: A rat cardiac myocardial cell line, H9C2, was exposed to CY metabolized by S9 fraction of rat liver homogenate mixed with co-factors (CYS9). The concentration of CY in this experiment was determined based on values measured in blood samples from patients receiving CY in high doses. The degree of cytotoxicity was then evaluated by MTT assay, LDH release, production of reactive oxygen species (ROS), and incidence of apoptosis. We also investigated how the myocardial cellular effects of CYS9 were modified by acrolein scavenger N-acetylcysteine (NAC), antioxidant isorhamnetin (ISO), and ƒÀ-ionone (BIO) a CYP inhibitor. Quantifying CY and CY metabolites, using liquid chromatography coupled with electrospray tandem mass spectrometry (LC/MS/MS), we assayed culture supernatants of CYS9 with or without PCA and also tested with HCY, CEPM and acrolein. In addition, we observed the cytotoxicity of CYS9 and the protective effects of PCA using a live-cell imaging system. Finally, using leukemia cell lines, we confirmed whether each of the above PCAs inhibits the antileukemic effects of CYS9.Results: The peak average concentration of CY in three patients receiving high-dose CY therapy was 257 ± 46 ƒÊM. After 250 ƒÊM of CY was metabolized in S9 mix for 2 h, the concentration of HCY was 17.6 ± 4.3, CEPM 26.6 ± 5.3 ƒÊM, and acrolein 30.8 ± 1.3 ƒÊM. Assay results for MTT and LDH release showed that treatment with CY (500 ƒÊM) did not cause cytotoxicity. However, CYS9 (CY 250 ƒÊM) exhibited myocardial cytotoxicity. NAC, ISO, and BIO all inhibited CYS9-induced cytotoxicity (Fig.1). When treated with ISO or BIO, metabolism of CY was significantly inhibited. Pre-treatment with NAC, however, did not inhibit the metabolism of CY. Compared to control samples, we observed no difference in HCY, a significant increase of CEPM, and a significant decrease of acrolein (Fig.2). Furthermore, NAC pre-treatment did not affect intracellular ROS levels produced by CYS9. Live-cell imaging also confirmed that CYS9 induced acute cytotoxicity, which was inhibited by NAC. Finally, NAC did not inhibit the CYS9-induced antileukemic effect.Discussion: According to results from LC/MS/MS, we ascertained that, when metabolized in vitro by S9, CY would produce similar levels of metabolic substance concentrations as in vivo. When H9C2 was pre-treated with NAC, ISO, and BIO, each inhibited cell damage by CYS9. When treated with ISO and BIO, we observed that CY metabolism itself was inhibited. This result suggests that cardiac cell damage may depend on individual differences in CY metabolic capacity. As preventative drugs, however, both BIO and ISO seem likely to interfere with antitumor effects and are thought to be unsuitable.NAC pre-treatment did not inhibit CY metabolism: compared to control samples, there was no difference in HCY concentration, but there was a significant increase in CEPM concentration. Less acrolein, however, was present after exposure to NAC. The increase in CEPM suggests that scavenging of acrolein by NAC prevents inhibition of ALDH1; in the absence of acrolein, AldoCY is metabolized to CEPM. NAC attenuates high-dose CY induced cardiotoxicity through a mechanism related to its ability to decrease of acrolein. [Display omitted] [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Erratum to: Histamine release from rat mast cells induced by the metabolic activation of drugs of abuse into free radicals

The metabolic activation of morphine, cocaine and methadone into free radicals could have pathophysiological relevance in the organic injuries of drug addiction.Isolated purified rat serosal mast cells were incubated with morphine, cocaine and methadone (10(-7) M-10(-4) M) with oxidative enzymes (prostaglandin-H-synthetase, 25 mU; rat liver homogenate fraction S 10-mix, 400 microl), and with the drugs of abuse in the presence of oxidative enzymes. Histamine and lactate dehydrogenase (LDH) were analysed with a fluorimetric and spectrophotometric assay, respectively; the generation of malonyldialdehyde (MDA) was measured by a spectrophotometric assay.The release of mast cell histamine and the generation of MDA are present only when mast cells were incubated with the drugs of abuse in the presence of oxidative enzymes. This release was dependent on the concentration of the drug in question and showed a maximum value at 10(-4) M. Moreover, in parallel experiments we demonstrated that, under the same experimental conditions, the release of LDH was always less than 20% of the total, suggesting that this effect is due to a selective exocytotic process. Histamine release and MDA generation were abated by the free radical scavengers: reduced glutathione, 10(-4) M GSH and alpha-tocopherol, 10(-4) M and by the spin trapper 5.5-dimethyl-1-pyrroline-N-oxide, 10(-4) M DMPO. The light and electron microscopic features are consistent with exocytotic secretion in the cases of morphine and methadone and with cell lysis in the case of cocaine.These results suggest that morphine, cocaine and methadone are activated into free radicals which produce membrane lipid perturbation and histamine release, suggesting that a massive release of mast cell histamine could be an additional risk factor in heroin and cocaine overdoses.

The metabolism of thioamides by the supernatant fraction of rat liver homogenate.

Abstract α-Phenyl-α-(2-pyridyl) thioacetamide (SC 15396; antigastrin) is metabolized by the supernatant fraction of rat liver homogenate to give two metabolites. The major metabolite has been identified as α-phenyl-α-(2-pyridyl)acetonitrile. Three other thioamides, analogues of SC 15396 also give the corresponding nitriles in these conditions.

The stimulation of strychnine metabolism in rats by some anticonvulsant compounds

Abstract The metabolism of a series of 4-acetyl-1-naphthyl ethers has been examined in rats and in fractions of rat liver homogenate. The administration of the compounds to rats increases the rate of strychnine metabolism when the compounds used are metabolised by the microsomal fraction of liver homogenate.

Cytotoxicity evaluation of reactive metabolites using rat liver homogenate microsome-encapsulated alginate gel microbeads

We present an improved cytotoxicity test for reactive metabolites, in which the S9 microsomal fraction of rat liver homogenate is encapsulated in alginate gel microbeads to avoid cytotoxic effects of S9-self-generated toxicants, microsomal lipid peroxides. The S9-encapsulated gel microbeads were prepared by a coaxial two-fluid nozzle and surfaces of the microbeads were coated with poly-L-lysine (PLL). Although the initial metabolic rate of the S9-encapsulated gel microbeads was about 20% slower than that of bare S9, the microbeads prevented the leakage of microsomal lipid peroxides thanks to the dense alginate and PLL polymer networks. In fact, the half maximal effective concentration of the indirect mutagen cyclophosphamide on NIH3T3 cells in the presence of the S9-encapsulated gel microbeads was about 5 times higher than that in the presence of bare S9. Use of the S9-encapsulated gel microbeads enabled the more accurate evaluation of the cytotoxicity of the reactive metabolites without the S9-based cytotoxicity.

Vesicular distribution of Secretory Pathway Ca2+-ATPase isoform 1 and a role in manganese detoxification in liver-derived polarized cells

Manganese is a trace element that is an essential co-factor in many enzymes critical to diverse biological pathways. However, excess Mn(2+) leads to neurotoxicity, with psychiatric and motor dysfunction resembling parkinsonism. The liver is the main organ for Mn(2+) detoxification by excretion into bile. Although many pathways of cellular Mn(2+) uptake have been established, efflux mechanisms remain essentially undefined. In this study, we evaluated a potential role in Mn(2+) detoxification by the Secretory Pathway Ca(2+), Mn(2+)-ATPase in rat liver and a liver-derived cell model WIF-B that polarizes to distinct bile canalicular and sinusoidal domains in culture. Of two known isoforms, only secretory pathway Ca(2+)-ATPase isoform 1 (SPCA1) was expressed in liver and WIF-B cells. As previously observed in non-polarized cells, SPCA1 showed overlapping distribution with TGN38, consistent with Golgi/TGN localization. However, a prominent novel localization of SPCA1 to an endosomal population close to, but not on the basolateral membrane was also observed. This was confirmed by fractionation of rat liver homogenates which revealed dual distribution of SPCA1 to the Golgi/TGN and a fraction that included the early endosomal marker, EEA1. We suggest that this novel pool of endosomes may serve to sequester Mn(2+) as it enters from the sinusoidal/basolateral domains. Isoform-specific partial knockdown of SPCA1 delayed cell growth and formation of canalicular domain by about 30% and diminished viability upon exposure to Mn(2+). Conversely, overexpression of SPCA1 in HEK 293T cells conferred tolerance to Mn(2+) toxicity. Taken together, our findings suggest a role for SPCA1 in Mn(2+) detoxification in liver.

In vitro mutagenicity of anti-inflammatory parsalmide analogues PA7, PA10, and PA31 triggered by biotransformation into hydroxy derivatives

In this study, the mutagenicity of the anti-inflammatory parsalmide [5-amino- N-butyl-2-(2-propynyloxy)-benzamide] analogues PA7 [5-amino- N-butyl-2-cyclohexyloxy-benzamide], PA10 [5-amino- N-butyl-2-phenoxy-benzamide] and PA31 [5-amino- N-butyl-2-( p-tolyloxy)-benzamide] was determined by an Ames Salmonella assay. The experiments were performed by preincubating the compounds in the absence and presence of a post-mitochondrial fraction (S9) of rat liver homogenate from phenobarbital/β-naphtoflavone treated rats. No mutagenic effect was observed after direct testing (no S9 added) in Salmonella typhymurium strains TA98, TA100, TA102, TA1535 and TA1537. However, in the presence of S9, the test substances triggered mutagenic responses in strains TA100 and TA98. PA31 presented the strongest mutagenic potential. The reversion rates in the presence of PA31 were about 2–19 fold higher than spontaneous mutation rates. In the presence of PA7, the reversion increased 2–14-fold over spontaneous rates. While PA10 showed a relatively mild mutagenic potential, as the number of revertants did not exceed 2.5 times the number of spontaneous mutations. Mass spectrometric analysis of the in vitro biotransformation showed that S9 converted (%), regioselectively, PA7 (19%), PA10 (7%) and PA31 (12%) into hydroxy-derivatives.

Structure-activity relationship of oxadiazoles and allylic structures in the Ames test: an industry screening approach.

In recent years genotoxicity testing has become more and more important in the process of early screening for potential development compounds. In the case that a pharmacologically interesting structure is found to be positive in an in vitro mutagenicity test a straightforward approach starts by sorting out what substructure is responsible for the activity observed in the test. The Ames test is a rapid, convenient test system which has been effectively used in structure-activity relationship studies for mutagenicity, since it can rapidly establish differences in the mutagenic action of isomers and chemical analogs. The lead compound with a benzodiazepine-like structure and close analogs exhibited weak, but unequivocal positive effects in the Ames test (strains TA1535 and TA 100) after metabolic activation by rat liver homogenate fraction (S9). To identify substances within this class of compounds devoid of mutagenic liability an extensive structure-activity investigation was undertaken. More than 50 compounds were tested in the two critical bacterial strains, using a standard plate incorporation and a preincubation modification. It quickly became evident that the benzodiazepine structure was not involved. First hints that the allyl side chain were responsible for the Ames activity had to be refined in a more complex, but clear-cut structure-activity relationship during the course of the experiments. It was shown that all compounds with an allyl side chain, independent of the heterocycle, but surprisingly also all compounds with a specific arrangement of the heteroatoms in the oxadiazole ring, showed positive effects in at least one strain. Based on these investigations it was possible to select pharmacologically active structures without mutagenic liability.

Localization of cytosolic NADP-dependent isocitrate dehydrogenase in the peroxisomes of rat liver cells: biochemical and immunocytochemical studies.

Two types of NADP-dependent isocitrate dehydrogenases (ICDs) have been reported: mitochondrial (ICD1) and cytosolic (ICD2). The C-terminal amino acid sequence of ICD2 has a tripeptide peroxisome targeting signal 1 sequence (PTS1). After differential centrifugation of the postnuclear fraction of rat liver homogenate, approximately 75% of ICD activity was found in the cytosolic fraction. To elucidate the true localization of ICD2 in rat hepatocytes, we analyzed the distribution of ICD activity and immunoreactivity in fractions isolated by Nycodenz gradient centrifugation and immunocytochemical localization of ICD2 antigenic sites in the cells. On Nycodenz gradient centrifugation of the light mitochondrial fraction, ICD2 activity was distributed in the fractions in which activity of catalase, a peroxisomal marker, was also detected, but a low level of activity was also detected in the fractions containing activity for succinate cytochrome C reductase (a mitochondrial marker) and acid phosphatase (a lysosomal marker). We have purified ICD2 from rat liver homogenate and raised a specific antibody to the enzyme. On SDS-PAGE, a single band with a molecular mass of 47 kD was observed, and on immunoblotting analysis of rat liver homogenate a single signal was detected. Double staining of catalase and ICD2 in rat liver revealed co-localization of both enzymes in the same cytoplasmic granules. Immunoelectron microscopy revealed gold particles with antigenic sites of ICD2 present mainly in peroxisomes. The results clearly indicated that ICD2 is a peroxisomal enzyme in rat hepatocytes. ICD2 has been regarded as a cytosolic enzyme, probably because the enzyme easily leaks out of peroxisomes during homogenization. (J Histochem Cytochem 49:1123-1131, 2001)

Further characterization of rat dihydroceramide desaturase: Tissue distribution, subcellular localization, and substrate specificity

The introduction of the double bond in the sphingoid backbone of sphingolipids occurs at the level of dihydroceramide via an NADPH-dependent desaturase, as discovered in permeabilized rat hepatocytes. In the rat, the enzyme activity, which has now been further characterized, appeared to be mostly enriched in liver and Harderian gland. By means of subcellular fractionation of rat liver homogenates and density gradient separation of microsomal fractions, the desaturase was localized to the endoplasmic reticulum. Various detergents were inhibitory to the enzyme, and maximal activities were obtained in the presence of NADPH and when the substrate was complexed to albumin. In the presence of albumin, the chain length of the fatty acid of the truncated dihydroceramides hardly affected the activity. Finally, in view of a likely evolutionary relationship between desaturases and hydroxylases, the formation of hydroxylated intermediates was analyzed. No evidence for their presence was found under our assay conditions.

P53 and Tumor Necrosis Factor α Regulate the Expression of a Mitochondrial Chloride Channel Protein

A novel chloride intracellular channel (CLIC) gene, clone mc3s5/mtCLIC, has been identified from differential display analysis of differentiating mouse keratinocytes from p53+/+ and p53-/- mice. The 4.2-kilobase pair cDNA contains an open reading frame of 762 base pairs encoding a 253-amino acid protein with two putative transmembrane domains. mc3s5/mtCLIC protein shares extensive homology with a family of intracellular organelle chloride channels but is the first shown to be differentially regulated. mc3s5/mtCLIC mRNA is expressed to the greatest extent in vivo in heart, lung, liver, kidney, and skin, with reduced levels in some organs from p53-/- mice. mc3s5/mtCLIC mRNA and protein are higher in p53+/+ compared with p53-/- basal keratinocytes in culture, and both increase in differentiating keratinocytes independent of genotype. Overexpression of p53 in keratinocytes induces mc3s5/mtCLIC mRNA and protein. Exogenous human recombinant tumor necrosis factor alpha also up-regulates mc3s5/mtCLIC mRNA and protein in keratinocytes. Subcellular fractionation of keratinocytes indicates that both the green fluorescent protein-mc3s5 fusion protein and the endogenous mc3s5/mtCLIC are localized to the cytoplasm and mitochondria. Similarly, mc3s5/mtCLIC was localized to mitochondria and cytoplasmic fractions of rat liver homogenates. Furthermore, mc3s5/mtCLIC colocalized with cytochrome oxidase in keratinocyte mitochondria by immunofluorescence and was also detected in the cytoplasmic compartment. Sucrose gradient-purified mitochondria from rat liver confirmed this mitochondrial localization. This represents the first report of localization of a CLIC type chloride channel in mitochondria and the first indication that expression of an organellular chloride channel can be regulated by p53 and tumor necrosis factor alpha.

Studies on the stability of trans-α-acetoxytamoxifen in Sprague–Dawley female rat liver slices, homogenate and subcellular fractions including microsomes and mitochondria

This work examined the stability of trans α-acetoxytamoxifen in Krebs–Henseleit buffer (pH 7.4), in the presence of Sprague–Dawley female rat liver slices, rat liver homogenate and hepatic subcellular fractions, including microsomes and mitochondria, at pH 7.4 and at 37°C over 300 min. The rate of hydrolysis was determined using high-performance liquid chromatography, and degradation profiles were obtained from which the rate and order of degradation were both evaluated. By applying zero-, first-, second- and third-order models of drug disappearance and the generation of by-products, first- and second-order appeared to produce the best fit. trans-α-Acetoxytamoxifen degraded rapidly in buffer and more slowly in the biological systems, probably due to the fact that the agent partitions into the hydrophobic component of the biological tissue and hence degrades at a much slower rate. The principal degradation products were trans-α-hydroxytamoxifen and, to a lesser extent, cis-α-hydroxytamoxifen. Another peak could not be identified. The production of trans-α-hydroxytamoxifen was enhanced in the presence of biological enzymes, whereas the concentration of the cis isomer remains relatively constant in buffer only (pH 7.4) and in the presence of biological enzymes. Therefore, the formation of identical adducts with DNA is consistent, because it has been shown that α-acetoxytamoxifen breaks down to form α-hydroxytamoxifen in vitro. The percentage of trans-α-acetoxytamoxifen remaining after 300 min was 40% in mitochondria and 32% in homogenate. The half-life ( t 1/2) was calculated for each condition by applying zero-, first- and second-order rate kinetics.

Enzymic formation of riboflavin 4',5'-cyclic phosphate from FAD: evidence for a specific low-Km FMN cyclase in rat liver1.

An enzyme activity splitting FAD to AMP and riboflavin 4',5'-cyclic phosphate (4',5'-cFMN), with a Km of 6-8 microM, was partially purified from the cytosolic fraction of rat liver homogenates. 4', 5'-cFMN was characterized by enzyme, HPLC, UV-visible and NMR spectroscopic analyses. The data suggest that a novel enzyme, tentatively named FAD-AMP lyase (cyclizing) or FMN cyclase, is involved. Also, 4',5'-cFMN was hydrolysed to 5'-FMN by a rat liver cyclic phosphodiesterase. The results indicate a novel enzymic pathway for flavins in mammals, and support the biological relevance of 4',5'-cFMN, perhaps as a flavocoenzyme or a regulatory signal.

Histamine release from rat mast cells induced by the metabolic activation of drugs of abuse into free radicals.

The metabolic activation of morphine, cocaine and methadone into free radicals could have pathophysiological relevance in the organic injuries of drug addiction. Isolated purified rat serosal mast cells were incubated with morphine, cocaine and methadone (10(-7) M-10(-4) M) with oxidative enzymes (prostaglandin-H-synthetase, 25 mU; rat liver homogenate fraction S 10-mix, 400 microl), and with the drugs of abuse in the presence of oxidative enzymes. Histamine and lactate dehydrogenase (LDH) were analysed with a fluorimetric and spectrophotometric assay, respectively; the generation of malonyldialdehyde (MDA) was measured by a spectrophotometric assay. The release of mast cell histamine and the generation of MDA are present only when mast cells were incubated with the drugs of abuse in the presence of oxidative enzymes. This release was dependent on the concentration of the drug in question and showed a maximum value at 10(-4) M. Moreover, in parallel experiments we demonstrated that, under the same experimental conditions, the release of LDH was always less than 20% of the total, suggesting that this effect is due to a selective exocytotic process. Histamine release and MDA generation were abated by the free radical scavengers: reduced glutathione, 10(-4) M GSH and alpha-tocopherol, 10(-4) M and by the spin trapper 5.5-dimethyl-1-pyrroline-N-oxide, 10(-4) M DMPO. The light and electron microscopic features are consistent with exocytotic secretion in the cases of morphine and methadone and with cell lysis in the case of cocaine. These results suggest that morphine, cocaine and methadone are activated into free radicals which produce membrane lipid perturbation and histamine release, suggesting that a massive release of mast cell histamine could be an additional risk factor in heroin and cocaine overdoses.

A comparative analysis of mutagenic activities of air samples collected from Riyadh before, during and after the Gulf War

Samples of air particulates were collected from the city of Riyadh in 1988 before the Gulf War, a few months after the torching of oil wells in Kuwait during 1991 and several months (in 1992) after the burning oil wells were fully capped. The air samples were collected on fibre glass filters (GF-A, pore size 1.6 mum) by using a RADECO air sampler. Filters were shredded, soaked and sonicated in acetone to dissolve the organic contents. The solid residues obtained after evaporation of acetone were redissolved in 5 ml volumes of fresh acetone and tested for mutagenic activity in the Ames Salmonella assay using the tester strain TA98 for histidine reversion (Ames 1979, Maron and Ames 1983). The mutagenicity assay was performed with and without metabolic activation provided by the S-9 fraction of an Aroclor-induced rat liver homogenate and co-factors. The results of this study showed that the pre-war samples of air particulates from Riyadh induced 11 histidine revertants/10 m3 of air analyzed, while the 1991 samples (during war) induced 40-50 revertants/10 m3 of air (4-5 times higher). However, the number of histidine revertants induced by the post-war (1992) samples came down to 10-11/10 m3 of air. These data, thus, strongly suggested that there was an increase in air particulate mutagenicity due to the atmospheric pollution of Gulf areas caused by the burning oil wells in Kuwait. This notion is supported by the fact that the mutagenic activity of air samples collected months after the burning oil wells were capped assumed the pre-war level. In view of the strong correlation between mutagenicity and carcinogenicity (McCann and Ames 1976) it would be expected that air pollution with mutagenic agents enhanced by the Gulf War would increase the carcinogenic risk for upper aerodigestive tracts in the exposed population in Kuwait and the surrounding areas. Such adverse health effects of mutagenic air pollutants generated by the burning oil wells in Kuwait should be assessed by proper epidemiologic studies.

FGF-2 antisense RNA encodes a nuclear protein with MutT-like antimutator activity

Bidirectional transcription of the basic fibroblast growth factor (FGF-2) gene gives rise to multiple polyadenylated sense mRNAs and a unique 1.5 kb antisense transcript (FGF-AS) which is complementary to the 3′-untranslated region of the FGF-2 mRNA. The rat FGF-AS cDNA encodes a novel 35 kDa nuclear protein (GFG) with homology to the MutT family of antimutator NTPases. Antibodies against the deduced amino acid sequence of GFG detected intense immunoreactivity in the nuclei of adult rat hepatocytes. Subcellular fractionation and Western blotting confirmed the presence of a 35 kDa immunoreactive protein in the nuclear fraction and, to a lesser extent, in the mitochondrial fractions of rat liver homogenates. Recombinant GFG suppressed the spontaneous mutation rate of MutT-deficient E. coli in a complementation assay. In-frame deletion of the 53 amino acids encompassing the MutT domain eliminated this activity, confirming the catalytic function of this region in the FGF antisense gene product. These findings demonstrate for the first time that the FGF-AS transcript encodes a functional nuclear protein with MutT-related enzymatic activity.

Hepatic production of 1,5-anhydrofructose and 1,5-anhydroglucitol in rat by the third glycogenolytic pathway.

A unique anhydrohexulose, 1,5-anhydrofructose (1,5AnFru) has been detected in rat livers. Here we describe a microanalytical method for 1,5AnFru using GC/MS and report results on the distribution and production of 1,5 AnFru in rats. The highest levels of 1,5AnFru were found in the liver (0.43 microgram/g wet tissue) and appreciable amounts were detected in adrenal gland and spleen (0.12 microgram/g and 0.09 microgram/g, respectively). Other organs contained lower amounts while plasma contained virtually no detectable 1,5AnFru. We also demonstrated that 1,5AnFru is produced in the cytosol fraction of rat liver homogenate when an alpha-1,4-glucan or glycogen was added; 1,5AnFru was readily reduced to 1,5-anhydroglucitol with NADPH or at a reduced efficiency with NADH in the presence of a Mono Q chromatographic fraction obtained from the same cytosol preparation. Based on these results, we propose the existence of a third degradation pathway, in addition to the phosphorolytic and hydrolytic reaction sequences, from glycogen to 1,5-anhydroglucitol via 1,5AnFru in mammals. However, the physiological significance of 1,5AnFru and this putative minor glycogenolytic pathway in mammals remains obscure.

Restricted Uptake of Dietary Coenzyme Q Is in Contrast to the Unrestricted Uptake of α-Tocopherol into Rat Organs and Cells

The dietary uptake of α-tocopherol and coenzyme Q was investigated in rats. Rats were fed diets supplemented with α-tocopherol or coenzyme Q10 (1 g/kg diet) or an unsupplemented control diet. In control rat tissues, the content of coenzyme Q was 4–11 times higher than that of α-tocopherol, but in plasma, the ratio was reversed. Among the subcellular fractions of rat liver homogenate, Golgi vesicles and lysosomes had the highest α-tocopherol concentration, and high concentrations of coenzyme Q were observed in the outer and inner mitochondrial membranes as well as in lysosomes, Golgi vesicles and plasma membranes. The uptake of α-tocopherol into the liver and plasma reached a maximal level after only 2 d of supplementation, whereas in the kidney, heart, muscle and brain, the levels continued to increase throughout the 6-wk treatment period. In contrast, dietary coenzyme Q was taken up into the liver and plasma only, and not into the other organs. This lipid appeared mainly in the Golgi system, whereas α-tocopherol exhibited a more general cellular distribution. The decay of the supplied α-tocopherol was slow in the various organs, but the disappearance of coenzyme Q was rapid from both liver and plasma. Pretreatment of rats with α-tocopherol increased the levels of both endogenous and exogenous coenzyme Q in the liver and plasma. These results demonstrate that the uptake of α-tocopherol from the diet is an extensive and general phenomenon at both the tissue and cellular levels, in contrast to the selective and restricted uptake of coenzyme Q.