Validity of mutagenic activity as an indicator of river water pollution.

OBJECTIVES: To investigate if mutagenicity could be expressed by known water pollution indicators, we determined the mutagenic activity of blue rayon extracts from sampled river water with the Ames test utilizing new strains of bacteria, and compared the results with those of known indicators of water pollution. METHODS: Water samples were collected by the blue rayon adsorption method at sixteen sites in six rivers in the North Kyushu district. The Assay of mutagenicity was carried out using the Ames test. The test strains wereSalmonella typhimurium TA100, YG1024, YG1041 and YG1042. B(a)P, Trp-P-1 and Trp-P-2 were quantified by HPLC. Determinations of SS, BOD, COD, T-N, T-P, DOC, and A(260)/DOC were performed. RESULTS: The extracts from five sampling sites showed higher mutagenicity toward strain YG1024 with or without S9mix, and the extracts from two of these five sites showed higher mutagenicity toward strain YG1041 with and without S9mix. However, the water pollution indicators did not show specific trends that were consistent with the mutagenic activity. CONCLUSIONS: Since the mutagenic activity of river water could not be predicted using known water pollution indicators, we recommend that biological examinations such as mutagenicity tests be added to the indicators that are currently in use.

The mutagenicity of amino-derivatives of diphenyl ether herbicides in new Salmonella typhimurium YG tester strains.

In this study, we examined the mutagenicity of four diphenyl ether herbicides and their amino derivatives in Salmonella typhimurium TA tester strains and YG tester strains. YG tester strains have been newly developed for sensitive detection of specific chemicals. S. typhimurium YG 1021, YG 1024, YG 1026 and YG 1029 strains are sensitive to mutagenic nitoroarenes and hydroxyamines. S. typhimurium YG 3003 is a strain that is sensitive to some oxidative mutagens. And S. typhimurium YG 7108 is useful for detection of mutagenic alkylating agents. As a result, each amino derivative of diphenyl ether herbicides is more mutagenic than its parent herbicide in S. typhimurium TA and YG tester strains with metabolic activation by S9 mixture. Moreover, S. typhimurium YG tester strains are more useful for highly sensitive detection of mutagens than S. typhimurium TA tester strains. We also examined the production of amino derivatives in a water environment from parent herbicides. It was clear that diphenyl ether herbicides rapidly transform to amino derivatives in a water environment.

Exogenously added alkylmethylglycerophosphocholine and alkylmethylcarbamylglycerophosphocholine accumulate in plasma membranes more than in intracellular membranes of rabbit platelets.

We found that extracellular addition of 2% bovine serum albumin (BSA) to a suspension of rabbit platelets after stimulation with platelet-activating factor resulted in a biphasic extraction of [3H]1-O-alkyl-2-O-methyl (or 2-O-methylcarbamyl)-sn-glycero-3-phosphocholine. A fast phase of extraction of the phospholipid probe by BSA was found to be mainly due to removal of the probe remaining in an outer layer of platelet plasma membrane, whereas a second phase of extraction of the probe by BSA was mostly attributed to redistribution of the probe which had been flipped across the plasma membrane. On the basis of analysis of the biphasic extraction by BSA of 1-O-alkyl-2-O-methyl (or methylcarbamyl)-sn-glycero-3-phosphocholine at various times after its addition, we suggested that the radioactive phospholipid accumulated in plasma membrane more than in intracellular membranes of rabbit platelets. In similar experiments with guinea-pig polymorphonuclear leukocytes, we observed a monophasic extraction of 1-O-alkyl-2-O-methyl (or methylcarbamyl)-sn-glycero-3-phosphocholine by BSA, indicating its unidirectional movement across the plasma membrane.

Exogenous phosphatidic acid with saturated short-chain fatty acyl groups induces superoxide anion release from guinea pig peritoneal polymorphonuclear leukocytes by three different mechanisms.

Treatment of suspensions of guinea pig peritoneal polymorphonuclear leukocytes (PMN) with four species of phosphatidate (PA) containing short-chain fatty acids induced sustained superoxide anion (O2-) production after a lag time. The rank order of efficiency of these PAs in triggering O2- production was PA8:0 [1,2-dioctanoyl-sn-glycerol-3-phosphate (GP)] > PA10:0 (1,2-didecanoyl-GP) > PA6:0 (1,2-dicaproyl-GP) > > PA12:0 (1,2-dilauroyl-GP). The O2- release from PMN stimulated with PA10:0 or PA12:0, but not with PA6:0 or PA8:0, was lowered by the addition of 1 mM extracellular Ca2+. Studies with various inhibitors showed that the mechanism of multiphasic O2- production induced by PA8:0 depended on its concentration: 1 and 3 microM PA8:0 induced O2- production constantly after a lag time through a protein kinase-dependent mechanism that was inhibited by 100 nM staurosporine. With concentrations of PA of 10 microM or more, an additional mechanism that was independent of protein kinase became operative and predominant over the protein kinase-dependent one. This protein kinase-independent mechanism was inhibited selectively by 80 microM TMB-8. Concentrations of 30, 60 and 100 microM PA first elicited transient O2- production via another protein kinase-dependent mechanism that was more sensitive to H-7 than to staurosporine, and then sustained O2- production, mainly driven by the protein kinase-independent mechanism. Metabolism of exogenously added [14C]PA8:0 in intact PMN was examined in the presence and absence of propranolol. Results suggest that PA itself is more important rather than its degradation products such as diacylglycerol, in inducing O2- production via three different mechanisms described above.

Platelet-activating factor (PAF)-like phospholipids formed during peroxidation of phosphatidylcholines from different foodstuffs.

Previously, we reported that induction of peroxidation of synthetic phosphatidylcholines (PCs) containing a polyunsaturated fatty acid by Fe(2+)-EDTA in the presence of ascorbate resulted in the formation of four types of PCs with an sn-2-oxidatively fragmented acyl group, which had platelet-aggregating activity due to interaction with platelet-activating factor (PAF) receptors. These PCs were compounds with a short-chain monocarboxylate, omega-hydroxymonocarboxylate, dicarboxylate, and dicarboxylate semialdehyde residue, respectively. In this study, we investigated the PAF-like lipids formed during peroxidation of PCs from hen egg yolk, salmon roe, sea urchin eggs, and krill in an FeSO4/EDTA/ascorbate system. The platelet-aggregating activities of these oxidized PCs were all inhibited by FR-900452, an antagonist of PAF. The activity of oxidized krill PC, which was equivalent of 89.8 +/- 8.8 pmol 16:0-PAF/mumol of starting PC, was about 5 times those of oxidized PCs from salmon roe and sea urchin eggs, and about 50 times that of oxidized hen egg yolk PC. The PAF-like phospholipids that had different combinations of long-chain alkyl or acyl groups with one of the above four types of short-chain acyl groups were identified by gas chromatography-mass spectrometry. The results indicated that foodstuffs that are rich in 1-O-alkyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine are potential sources of compounds with high PAF-like activity formed by deleterious lipid peroxidation.

Platelet-activating factor and its structural analogues in the earthworm Eisenia foetida.

The earthworm Eisenia foetida was shown to contain large amounts of ether-containing phospholipids such as alkylacylglycerophosphocholine (61.3% of choline glycerophospholipids) and alkenylacylglycerophosphoethanolamine (66.0% of ethanolamine glycerophospholipids). We also found a substantial amount of ether-containing PAF-like lipid in this animal, its level being increased after the animal is injured. We showed evidence that this PAF-like lipid consists of PAF and PAF analogues containing short chain fatty acids other than acetic acid. Notably, a propionic acid-containing species but not PAF itself, is the most predominant species in this animal. We also confirmed that the earthworms contain enzyme activities involved in the synthesis of PAF and short chain fatty acid-containing PAF analogues. Interestingly, the acetyltransferase activity in earthworms is resistant to high concentrations of the substrate lysophospholipid. Thus, both the structure of the PAF-like lipid and the properties of the enzymes involved in the PAF-like lipid metabolism in the earthworms are somewhat different from those in mammalian tissues.

Platelet-aggregating effects of platelet-activating factor-like phospholipids formed by oxidation of phosphatidylcholines containing an sn-2-polyunsaturated fatty acyl group.

Previously, we reported the formation of four kinds of phosphatidylcholines (PC) with a short-chain monocarboxylate, dicarboxylate, dicarboxylate semialdehyde or omega-hydroxymonocarboxylate group by oxidation of PCs containing polyunsaturated fatty acid (PUFA) in an FeSO4/ascorbate/EDTA system. In this study, we identified these novel phospholipids by GC-MS as oxidation products of two alkyl ether-linked PCs, 1-O-hexadecyl-2-docosahexaenoyl and 1-O-hexadecyl-2-arachidonoyl-sn-glycero-3- phosphocholine (GPC). The sn-2-acyl moieties of oxidatively fragmented PCs derived from PCs containing docosahexaenoate were one methylene unit shorter than those detected as major oxidation products of PCs containing arachidonate. The platelet-aggregations induced by the oxidized PCs were all inhibited by FR-900452, an antagonist of platelet activating factor (PAF). The PAF-like activity of oxidized 1-O-hexadecyl-2-docosahexaenoyl-GPC, which was equivalent of 1372 +/- 262 pmol 16:0-PAF/mumol starting PC, was 5 times that of oxidized 1-O-hexadecyl-2-arachidonoyl-GPC and 150 times that of oxidized 1-palmitoyl-2-docosahexaenoyl-GPC, suggesting that both an sn-1-alkyl ether linkage and an sn-2-acyl group with a short chain length are important structural requirements for induction of platelet aggregation. These possibilities were confirmed by experiments on the platelet-aggregating activities of synthetic PAF-like compounds. Quantitative measurements by GC-MS of PAF-like phospholipids formed by lipid peroxidation and the activities of synthetic PAF-like phospholipids, suggested that the activities of most oxidized PCs containing PUFA were ascribable to those of PCs with an sn-2-short-chain monocarboxylate group.

Formation of platelet-activating factor-like phospholipids by Fe2+/ascorbate/EDTA-induced lipid peroxidation.

We have identified novel phospholipids together with platelet-activating factor and its 1-acyl analogues in purified fractions from a bovine brain lipid extract. These novel compounds were phospholipids with an sn-2-short-chain monocarboxylyl, dicarboxylyl or omega-hydroxymonocarboxylyl group. The profiles of these three types of phospholipids suggest that they were formed by lipid peroxidation. To examine this possibility, we peroxidized synthetic phosphatidylcholines (PC) with an sn-2-polyunsaturated fatty acyl group and PC from bovine brain, with Fe2+/ascorbate/EDTA, and analyzed the secondary degradation products retaining a glycerol backbone by fast atom bombardment-mass spectrometry and GC-MS. Results showed the formation of four kinds of PC with a short-chain monocarboxylate, dicarboxylate, dicarboxylate semialdehyde or omega-hydroxymonocarboxylate moiety. The chain lengths of these PC were related to the position of the double bond vicinal to the esterified carbonyl group in the sn-2-long-chain acyl moiety of the parent PC. The molecular heterogeneity of secondary products formed by the oxidative degradation of bovine brain PC resembled those of the unique phospholipids that we previously detected in the fractions with platelet-activating factor-like activity purified from a bovine brain lipid extract, although the former lacked the species with an acetyl group. These results suggest that all the novel phospholipids with a short-chain acyl group in the brain lipid extract except that with an acetyl group were produced by lipid peroxidation.

Formation of PAF-like compounds by peroxidation of phospholipids from bovine brain.

This short review describes the identification by mass spectrometry of phospholipids having an sn-2-short-chain monocarboxylate, dicarboxylate or omega-hydroxycarboxylate group in a fraction with PAF-like activity from a bovine brain lipid extract. The similar molecular heterogeneities of these unique phospholipids suggest that they are formed via a common mechanism, possibly peroxidation of membrane phosphatidylcholines. This possibility is supported by the finding that these PAF-like compounds were actually formed during peroxidation of synthetic phosphatidylcholines induced by Fe2+/ascorbate/EDTA.

Possible influence of lysophospholipase on the production of 1-acyl-2-acetylglycerophosphocholine in macrophages.

The rate of production of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF) and 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (acylPAF) was measured in macrophages following the incorporation of [3H]acetate. Upon activation by A23187, guinea pig alveolar macrophages incorporated [3H]acetate into PAF, but a little radioactivity was found in acylPAF. However, labeling of acylPAF and PAF with [3H]acetate was greatly enhanced in A23187-stimulated alveolar macrophages that had been pretreated with phenylmethanesulphonyl fluoride (PMSF). [3H]PAF was predominantly converted to 1-[3H]alkyl-2-acyl glycerophosphocholine, but [14C]acylPAF rapidly hydrolyzed to 14C-labeled free fatty acid by the incubation with lysates prepared from macrophages. The deacetylation of [14C]acylPAF and [3H]PAF by acetylhydrolase and also the hydrolysis of [14C]lysoPC by lysophospholipase were strongly inhibited in macrophages that had been pretreated with PMSF, while PMSF failed to inhibit the activities of acetyltransferase and acyltransferase. The relative proportions of PAF and acylPAF were quite different in different types of cells. In contrast to alveolar macrophages, peritoneal macrophages, neutrophils and spleen cells from guinea pigs incorporated 2-4 times more [3H]acetate into acylPAF than into PAF. The presence of high levels of acylPAF in peritoneal macrophages was confirmed by GLC-MS analysis. The activities of lysophospholipase, acetylhydrolase and acetyltransferase were measured in alveolar and peritoneal macrophages to determine whether the preferential formation of acylPAF as compared to PAF in peritoneal macrophages was due to differences in these activities between alveolar and peritoneal macrophages. The activity of acetylhydrolase of peritoneal macrophages was almost the same as that in alveolar macrophages. The activity of acetyltransferase in peritoneal macrophages was about half of that in alveolar macrophages. However, the activity of lysophospholipase in peritoneal macrophages was one-sixth of that in alveolar macrophages. These results suggest that lysophospholipase is one of the primary factors involved in the control of the production of acylPAF in activated cells, and that it acts by modulating the availability of lysoPC for the synthesis of acylPAF. Furthermore, high levels of activity of lysophospholipase allow the preferential formation of PAF, via the rapid hydrolysis of lysoPC which would act as a competitive inhibitor of the incorporation of acetate into lysoPAF.

Transbilayer movement and metabolic fate of ether-linked phosphatidic acid (1-O-Octadecyl-2-acetyl-sn-glycerol 3-phosphate) in guinea pig peritoneal polymorphonuclear leukocytes.

1-O-Octadecyl-2-acetyl-sn-glycerol 3-phosphate (octadecylacetyl-GP) and its deacetylation product were used as a model of phosphatidic acid and its lyso derivatives, respectively. The binding, transbilayer movement, and intermembranous transport, which should be related to its metabolism in guinea pig peritoneal polymorphonuclear leukocytes, were studied. The albumin extraction procedure (Tokumura, A., Tsutsumi, T., Yoshida, J., and Tsukatani, H. (1990) Biochim. Biophys. Acta 1044, 91-100) was used for studying the transbilayer movement of [3H]octadecylacetyl-GP. The binding, translocation, and metabolism of octadecylacetylglycerol, a dephosphorylated product of octadecylacetyl-GP, in polymorphonuclear leukocytes were also investigated for comparison. The translocation of octadecylacetyl-GP was dependent on temperature, but not on its concentration (in the range of 1-100 nM). The rate of translocation of octadecylacetyl-GP was much slower than that of octadecylacetylglycerol. Treatment of polymorphonuclear leukocytes with N-ethylmaleimide did not affect the translocation of octadecylacetyl-GP. These results suggest that the transbilayer movement of octadecylacetyl-GP is driven by a diffusion process, not by a carrier protein. From these findings, the process of translocation of octadecylacetyl-GP is concluded to be a rate-limiting step in its metabolic conversion to triglyceride, phosphatidylethanolamine, and phosphatidylcholine.

Quantitative analysis of platelet-activating factor in rat brain.

Age-related decrease of the platelet-activating factor (PAF) content in rat brain was shown by a convenient method consisting of solid extraction of lipids with a Sep-Pak C-18 cartridge, lipid separation by HPLC and bioassay on rabbit platelets. This method was sufficiently sensitive to allow measurement of PAF in a single brain, and the recovery of PAF was quite high throughout the procedure.

Antagonism of platelet-activating factor in isolated rat colon: possible mechanism.

The contractions of three different regions of rat colon in response to platelet-activating factor (PAF) were compared. The ascending colon was found to be the most responsive. The slow contraction of the ascending colon induced by PAF was dependent on external Ca2+. CV-3988, a structural analog of PAF, slowly induced irreversible inhibition of PAF-induced contraction, whereas FR-900452, which is structurally unrelated to PAF, caused rapid reversible inhibition of PAF-induced contraction. No inhibitory effects of CV-3988 were observed when the strip was washed with Tyrode's solution containing 1% bovine serum albumin (BSA). The results suggest that PAF and CV-3988 penetrate slowly into the outer half of the lipid bilayer of plasma membranes of cells in isolated rat colon, and then rapidly diffuse laterally to associate firmly with specific binding sites.

Lysophosphatidic acids induce contraction of rat isolated colon by two different mechanisms.

Lysophosphatidic acids (1-linoleoyl-, 1-linolenoyl, 1-arachidonoyl- and 1-O-hexadecyl-sn-glycero-3-phosphate) induced rapid contraction of rat isolated colon which was dependent on external Ca2+, 1-linolenoyl-lysophosphatidic acid having the greatest effect. The contraction induced by 1-linolenoyl-lysophosphatidic acid was reduced considerably by nifedipine and verapamil, but not by atropine or indomethacin. Phosphatidic acids with two short-chain acyl groups induced a small, atropine-sensitive contraction at 100 microM, but phosphatidic acids with two long-chain acyl groups were inactive. These results suggest that unlike phosphatidic acids, lysophosphatidic acids act directly on extracellular sites of the plasma membrane of smooth muscle cells in rat colon, mainly through activation of voltage-sensitive Ca2+ channels.

Identification of sn-2-omega-hydroxycarboxylate-containing phospholipids in a lipid extract from bovine brain.

Phospholipids having both a long-chain acyl (palmitoyl or stearoyl) and a short-chain hydroxycarboxylyl (C3-C9) residue were identified by GC-MS in a fraction with PAF-like activity from a bovine brain lipid extract. The hydroxyl group in the hydroxycarboxylate residue was determined to be at the omega-position by comparison of the mass spectra of the tert-butyl-dimethylsilyl derivatives of these compounds with those of synthetic hydroxybutyrate-containing phosphatidylcholines. The co-existence of short-chain hydroxycarboxylate-, monocarboxylate- and dicarboxylate-containing phospholipids in the bovine brain lipid extract suggested that these compounds were formed by peroxidation of membrane phospholipids, especially phosphatidylcholines.

Translocation of exogenous platelet-activating factor and its lyso-compound through plasma membranes is a rate-limiting step for their metabolic conversions into alkylacylglycerophosphocholines in rabbit platelets and guinea-pig leukocytes.

Platelets and leukocytes are known to degrade platelet-activating factor (PAF), a potential mediator of inflammation, to its lyso-derivative (lyso-PAF) and then convert this to 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholines. However, little is known about the mechanism of internalization of PAF and lyso-PAF, which is a prerequisite for their metabolism within the cells. In this work, the internalization of PAF and lyso-PAF by rabbit platelet and guinea-pig leukocyte plasma-membranes were examined by the washing method with bovine serum albumin. The rates of translocation of PAF and lyso-PAF across guinea-pig plasma membranes were significantly higher than those across rabbit platelets. In these cells, the translocation of PAF was found to be accelerated indirectly by activation of PAF receptors by a small portion of added PAF. Results suggest that a temperature-dependent diffusion process is involved in the internalization of these phospholipids. In both rabbit platelets and guinea-pig leukocytes, the translocation of PAF and lyso-PAF through the plasma membranes was shown to be rate-limiting for the metabolic conversion of these compounds to 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine.

Vitamin E. deficiency increases the synthesis of platelet-activating factor (PAF) in rat polymorphonuclear leucocytes.

Vitamin E deficiency was found to stimulate FMLP (N-formyl-L-methionyl-L-leucyl-L-phenylalanine)-induced biosynthesis of PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in polymorphonuclear leucocytes (PMN) from rat peritoneum. In three separate experiments each, the amounts of PAF synthesized during 6 min and 12 min incubation of PMN cells from control, vitamin E-supplemented, and vitamin E-deficient rats were 129-240, 131-227 and 248-354 pmol/10(6) cells, respectively. The activity of the acetyl-transferase, which transfers the acetyl moiety of [3H]acetyl-CoA to 2-lysoPAF (1-O-alkyl-sn-glycero-3-phosphocholine) to form [3H]PAF, was higher in PMN homogenates from vitamin E-deficient rats (2.28 +/- 0.07 nmol/min/mg protein) than in those from E-supplemented rats (1.06 +/- 0.10 nmol/min/mg protein). However, there was no difference between the two groups in the activity of acetylhydrolase (4.26 +/- 0.71 and 4.26 +/- 0.06 nmol/min/mg protein, respectively), measured as degradation of [3H]PAF to [3H]lysoPAF. In vitro addition of alpha-tocopherol did not inhibit the increased activity of acetyl-transferase in vitamin E-deficient rats, indicating that the enzyme in vitamin E-supplemented rats was not directly inhibited by alpha-tocopherol. The acetyl-transferases of the two groups showed similar Km values for acetyl-CoA, but different Vmax values (225 microM and 6.4 nmol/min/mg protein in vitamin E-deficient rats, and 216 microM and 3.6 nmol/min/mg protein in vitamin E-supplemented rats), suggesting that the enzyme was not activated but increased in amount in vitamin E deficiency.

Novel molecular analogues of phosphatidylcholines in a lipid extract from bovine brain: 1-long-chain acyl-2-short-chain acyl-sn-glycero-3-phosphocholines.

A vasodepressor phospholipid fraction named Depressor-IA from a bovine brain lipid extract was analyzed by capillary gas-liquid chromatography-mass spectrometry as tert-butyldimethylsilyl derivatives after hydrolysis with phospholipase C. Results show that Depressor-IA is a mixture of 3 platelet-activating factors and 17 1-acyl analogues. Three platelet-activating factors having sn-1-O-hexadecyl, octadecyl, and octadecenyl groups were suggested to account for the hypotensive activity of Depressor-IA, although the total amount of 1-acyl analogues of platelet-activating factor was much more than that of platelet-activating factor in the purified Depressor-IA. 1-Long-chain acyl-2-short-chain acyl-glycero-3-phosphocholines identified in Depressor-IA included novel molecular analogues having sn-2-propionyl, acryloyl, butyryl, valeryl, caproyl, and hepatonoyl groups.

Novel phospholipids with aliphatic dicarboxylic acid residues in a lipid extract from bovine brain.

A vasodepressor phospholipid fraction purified from a lipid extract of bovine brain was found to contain novel phospholipids with both a long-chain acyl group and an aliphatic dicarboxylic acid residue. This was shown by analyzing the fraction as tert-butyldimethylsilyl derivatives of glyceride by capillary GC-MS after hydrolysis with phospholipase C. Six molecular species with a palmitoyl group and an aliphatic dicarboxylate (chain length C4-C9), and two species with both a stearoyl group and a succinate or glutarate residue were detected.

Study of platelet activating factor and its antagonists on rat colon strip with a new method avoiding tachyphylaxis.

Platelet activating factor induced slow, but sustained contraction of isolated rat colon in a dose-dependent manner. The contraction persisted even when the strips were washed several times with Tyrode solution. However, the addition of high concentrations of bovine serum albumin to the washing solution led to the rapid relaxation of the strips to the basal level, possibly by removing platelet activating factor tightly bound to rat colon. These strips then responded normally to a second addition of platelet activating factor. Neither the cholinergic nervous system nor release of histamine, serotonin, prostaglandins or leukotriene D4 were significantly involved in the contraction induced by platelet activating factor. FR-900452 and CV-3988, recently found to be antagonists of platelet activating factor, selectively blocked the contraction of rat colon induced by the active phospholipid, indicating that it induced contraction by a direct stimulatory effect on the smooth muscle of rat colon in a receptor-mediated manner.