Superoxide Anion Radical Release Research Articles

Aptamer-enhanced the Ag(I) ion-3,3′,5,5′-tetramethylbenzdine catalytic system as a novel colorimetric biosensor for ultrasensitive and selective detection of paraquat

A facile and simple colorimetric biosensor was first established for paraquat (PQ) detection based on the aptamer-enhanced oxidation process of 3,3′,5,5′-tetramethylbenzidine (TMB) by Ag+. The study confirmed that the interaction of PQ-15 aptamer with Ag+ accelerates the electron transfer from the aptamer-Ag+ complex to dissolved oxygen, which enhances the release of superoxide anion radicals (O2̇−) and facilitates the catalytic oxidation of the chromogenic substrate. PQ-15 aptamer will preferentially bind to PQ molecules, resulting in no further enhancement of the catalytic activity of Ag+. Molecular docking results revealed that the PQ molecules are attached to the stem-loop region of the PQ-15 aptamer through σ-π conjugation interactions. The proposed method is simple that only contains Ag+ and corresponding aptamer. The limit of detection (LOD) of the constructed colorimetric biosensor for PQ detection was determined to be 16.5 μg·L-1, belowing the maximum residue limit in fruits and vegetables set by the EU. Moreover, the colorimetric biosensor showed excellent selectivity and anti-interference properties, which was validated for detecting PQ residues in several typical agricultural and water samples.

Chemical Constituents of Bryophytes: Structures and Biological Activity.

Comparatively little attention has been paid to the bryophytes for use in the human diet or medicine in spite of the presence of 23 000 species globally. Several hundred new compounds have been isolated from the liverworts (Marchantiophyta), and more than 40 new carbon skeletons of terpenoids and aromatic compounds were found. Most of the liverworts studied elaborate characteristic odiferous, pungent, and bitter-tasting compounds, of which many show antimicrobial, antifungal, antiviral, allergic contact dermatitis, cytotoxic, insecticidal, anti-HIV, plant growth regulatory, neurotrophic, NO production and superoxide anion radical release inhibitory, muscle relaxing, antiobesity, piscicidal, and nematocidal activities. The biological effects ascribed to the liverworts are mainly due to lipophilic sesqui- and diterpenoids, phenolic compounds, and polyketides, which are the principal constituents of their oil bodies. Some mosses and liverworts produce significant levels of vitamin B2 and tocopherols, as well as prostaglandin-like highly unsaturated fatty acids. The most characteristic chemical phenomenon of the liverworts is that most of the sesqui- and diterpenoids are enantiomers of those found in higher plants. In this review, the chemical constituents and potential medicinal uses of bryophytes are discussed.

Cytotoxic and Antiviral Compounds from Bryophytes and Inedible Fungi

Over several hundred new compounds have been isolated from the bryophytes and more than 40 new carbon skeletal terpenoids and aromatic compounds found in this class. Most of liverworts elaborate characteristic odiferous, pungent and bitter tasting compounds many of which show, antimicrobial, antifungal, antiviral, allergenic contact dermatitis, cytotoxic, insecticidal, anti-HIV, superoxide anion radical release, plant growth regulatory, neurotrophic, NO production inhibitory, muscle relaxing, antiobesity, piscicidal and nematocidal activity. Several inedible mushrooms produce spider female pheromones, strong antioxidant, or cytotoxic compounds. The present paper concerns with the isolation of terpenoids, aromatic compounds and acetogenins from several bryophytes and inedible fungi and their cytotoxic and antiviral activity.

Pungent and Bitter, Cytotoxic and Antiviral Terpenoids from Some Bryophytes and Inedible Fungi

Most liverworts elaborate characteristic odiferous, pungent and bitter tasting compounds many of which show antimicrobial, antifungal, antiviral, allergenic contact dermatitis, cytotoxic, insecticidal, anti-HIV, superoxide anion radical release, plant growth regulatory, neurotrophic, NO production inhibitory, muscle relaxant, antiobesity, piscicidal and nematocidal activities. Several inedible mushrooms produce female spider pheromones, strong antioxidant, and cytotoxic compounds. The present paper is concerned with the extraction and isolation of terpenoids from some bryophytes and inedible fungi and their pungent and bitter taste, and cytotoxic and antiviral activity.

The effects of cyclooxygenase and nitric oxide synthase inhibition on oxidative stress in isolated rat heart

Despite the widespread clinical use of cyclooxygenase (COX) inhibitors, dilemmas still exist about potential impact of these drugs on cardiovascular system. The present study was aimed to estimate the effects of different COX inhibitors (meloxicam, acetylsalicylic acid [ASA], and SC-560) on oxidative stress in isolated rat heart, with special focus on L-arginine/NO system. The hearts of male Wistar albino rats (total number n = 96, each group 12 rats, 8 weeks old, body mass 180-200 g) were retrogradely perfused according to the Langendorff technique at gradually increased perfusion pressure (40-120 cmH2O). After control experiments the hearts were perfused with the following drugs: 100 μmol/l ASA (Aspirin), alone or in combination with 30 μmol/l L-NAME, 0.3 μmol/l meloxicam (movalis) with or without 30 μmol/l L-NAME, 3 μmol/l meloxicam (alone or in combination with 30 μmol/l L-NAME), 30 μmol/l L-NAME, and administration of 0.25 μmol/l SC-560. In samples of coronary venous effluent the following oxidative stress markers were measured spectrophotometrically: index of lipid peroxidation (measured as thiobarbituric acid reactive substances [TBARS]), superoxide anion radical release (O2(-)), and hydrogen peroxide (H2O2). While ASA was found to have an adverse influence on redox balance in coronary circulation, and coronary perfusion, meloxicam and SC-560 do not negatively affect the intact model of the heart. Furthermore, all effects were modulated by NOS inhibition. It seems that interaction between COX and L-arginine/NO system truly exists in coronary circulation, and can be one of the possible causes for achieved effects. That means: those effects induced by different inhibitors of COX are modulated by subsequent inhibition of NOS.

Search for New Liverwort and Fern Constituents of Biological Interest

Over several hundred new compounds have been isolated from the liverworts and more than 40 new carbon skeletal terpenoids (1,2) and aromatic compounds (3) have been found in this class. The biological activity ascribed to liverworts is due mainly to lipophilic sesqui- and diterpenoids, phenolic compounds and acetogenins which are constituents of oil bodies. Most of liverworts elaborate characteristic odiferous, hot tasting and bitter compounds many of which show allergenic contact dermatitis, cytotoxicity, insecticide, anti-HIV, antimicrobial, antifungal and antiinfluenza, superoxide anion radical release, plant growth regulatory, neurotrophic, NO production inhibitory, muscle relaxing and antiobesity activity.[1 – 3] Several ferns produce pungent, bitter or sweet substances. Thelypteris hispidula and Blechnum fluviatile produce a large amount of polygodial, responsible for their pungency and its related drimanes. Elaphoglossum and Hymenophyllum species elaborate bitter styrene glucosides, and hemiterpene glucosides (4) with phenylacetic acid, respectively. [4 – 6]

Cellular Protection During Oxidative Stress: A Potential Role for D-ribose and Antioxidants

ABSTRACTA healthy cellular system involves the maintenance of an intracellular metabolic balance. Reactive oxygen species (ROS) are constantly produced as a normal product of cellular metabolism; however, during situations of cellular stress, these levels can increase dramatically with the potential to cause deleterious cellular structural and/or functional consequences. There is a significant elevation in these ROS following stressful situations, such as ischemia, hypoxia, high-intensity exercise, and in many diseases. To combat these ROS, neutralizing endogenous enzymes, as well as exogenous antioxidants, can aid in minimizing their potential untoward cellular effects. Exogenous reducing antioxidant agents, such as vitamin C and/or E, play a role in addressing these formed species; however, recent research has suggested that fruit seed extracts may provide additional cellular benefits beyond their antioxidant features. Furthermore, supplemental D-ribose enhances the recovery of high-energy phosphates following stress and appears to potentially offer additional benefits by reducing radical formation. Specifically, during periods of hypoxia/ischemia, supplemental D-ribose may play an inhibitory role in the breakdown of adenine nucleotides, influencing the subsequent formation of xanthine and uric acid compounds; and thereby affecting the release of superoxide anion radicals. The combination of D-ribose with reducing antioxidants may provide a more optimal state of cellular protection during and following times of oxidative stress.

Phytochemical and biological studies of bryophytes

The bryophytes contain the Marchantiophyta (liverworts), Bryophyta (mosses) and Anthocerotophyta (hornworts). Of these, the Marchantiophyta have a cellular oil body which produce a number of mono-, sesqui- and di-terpenoids, aromatic compounds like bibenzyl, bis-bibenzyls and acetogenins. Most sesqui- and di-terpenoids obtained from liverworts are enantiomers of those found in higher plants. Many of these compounds display a characteristic odor, and can have interesting biological activities. These include: allergenic contact dermatitis, antimicrobial, antifungal and antiviral, cytotoxic, insecticidal, insect antifeedant, superoxide anion radical release, 5-lipoxygenase, calmodulin, hyaluronidase, cyclooxygenase, DNA polymerase β, and α-glucosidase and NO production inhibitory, antioxidant, piscicidal, neurotrophic and muscle relaxing activities among others. Each liverwort biosynthesizes unique components, which are valuable for their chemotaxonomic classification. Typical chemical structures and biological activity of the selected liverwort constituents as well as the hemi- and total synthesis of some biologically active compounds are summarized.

Inhibition of Amyloid Peptide Fragment Aβ25–35 Fibrillogenesis and Toxicity by N‐Terminal β‐Amino Acid‐Containing Esapeptides: Is Taurine Moiety Essential for In Vivo Effects?

We report the synthesis and fibrillogenesis inhibiting activity of the new peptide derivatives 1-4, related to the pentapeptide Ac-LPFFD-NH(2) (iAβ5p), proposed by Soto and co-workers and widely recognized as one of the most active β-sheet breaker agents. The Aβ(25-35) fragment of the parent full-length Aβ(1-42) was used as fibrillogenesis model. The activity of peptide derivatives 1-4 was tested in vitro by thioflavin T binding assay, far UV CD spectroscopy, and scanning electron microscopy. Their ability to hinder the toxic effect of Aβ(25-35) in vivo was studied by monitoring the viability of human SH-SY5Y neuroblastoma cells and the prevention of superoxide anion radical release from BV2 microglial cells. The results point to a favourable role in the fibrillogenesis inhibitory activity of the sulphonamide junction for compounds 1 and 2, containing an N,N-dimethyltaurine and a taurine amino-terminal moiety, respectively. Furthermore, compounds 1 and 2 show a significant protective effect on cell viability, rescuing the cells from the toxicity exerted by Aβ(25-35) treatment.

Pre-anthesis high-temperature acclimation alleviates damage to the flag leaf caused by post-anthesis heat stress in wheat

The objective of this study was to investigate the effect of pre-anthesis high-temperature acclimation on leaf physiology of winter wheat in response to post-anthesis heat stress. The results showed that both pre- and post-anthesis heat stresses significantly depressed flag leaf photosynthesis and enhanced cell membrane peroxidation, as exemplified by increased O 2 − production rate and reduction in activities of antioxiditave enzymes. However, under post-anthesis heat stress, plants with pre-anthesis high-temperature acclimation (HH) showed much higher photosynthetic rates than those without pre-anthesis high-temperature acclimation (CH). Leaves of HH plants exhibited a higher Chl a/b ratio and lower chlorophyll/carotenoid ratio and superoxide anion radical release rate compared with those of the CH plants. In addition, antioxidant enzyme activities in HH plants were significantly higher than in CH. Coincidently, expressions of photosythesis-responsive gene encoding Rubisco activase B ( RcaB) and antioxidant enzyme-related genes encoding mitochondrial manganese superoxide dismutase ( Mn-SOD), chloroplastic Cu/Zn superoxide dismutase ( Cu/Zn-SOD), catalase ( CAT) and cytosolic glutathione reductase ( GR) were all up-regulated under HH, whereas a gene encoding a major chlorophyll a/b-binding protein ( Cab) was up-regulated by post-anthesis heat stress at 10 DAA, but was down-regulated at 13 DAA. The changes in the expression levels of the HH plants were more pronounced than those for the CH. Collectively, the results indicated that pre-anthesis high-temperature acclimation could effectively alleviate the photosynthetic and oxidative damage caused by post-anthesis heat stress in wheat flag leaves, which was partially attributable to modifications in the expression of the photosythesis-responsive and antioxidant enzymes-related genes.

Heterozygous deficiency of manganese superoxide dismutase results in severe lipid peroxidation and spontaneous apoptosis in murine myocardium in vivo

To circumvent the early lethality of manganese superoxide dismutase (SOD2)-deficient mice, we have used a skin-specific strategy with introduction of loxP sites flanking exon 3 of the SOD2 gene. To our surprise, when breeding a female keratin 14 Cre transgenic mouse to a SOD2 “floxed” male mouse, due to keratin 14 promoter-driven Cre expression in the oocytes, all offspring were heterozygous for SOD2. In sharp contrast to initial publications on SOD2 +/− mice, the herein reported mice on a mixed genetic background (C57BL/6 × 129/Ola) in their heterozygous state (SOD +/−) revealed distinct ultrastructural damage of the myocard, with swelling and disruption of mitochondria and accumulation of lipid droplets, increased nitrotyrosine formation, and lipid peroxidation as well as activation of apoptosis signaling pathways in the heart in vivo. Strikingly, and so far unreported, we found a substantial decrease in the activity of the cytosolic copper, zinc superoxide dismutase (SOD1) in the heart tissue of SOD2 +/− mice, suggesting that the breakdown of mitochondrial membranes in the heart of SOD2 +/− mice results in the enhanced release of superoxide anion radicals or derivatives thereof with subsequent inactivation of cytosolic SOD1. This model may be particularly suited to long-term studies on age-related heart failure as well as other age-related diseases and the polygenic base of tissue-specific responses to oxidative injury.

Fifty-hertz magnetic fields induce free radical formation in mouse bone marrow-derived promonocytes and macrophages

Our findings show a significant increase of free radical production after exposure to 50 Hz electromagnetic fields at a flux density of 1 mT to mouse bone marrow-derived (MBM) promonocytes and macrophages, indicating the cell-activating capacity of extremely low frequency magnetic fields (ELF-MF). We demonstrate that after exposure to ELF-MF mainly superoxide anion radicals were produced, both in MBM macrophages (33%) and also in their precursor cells (24%). To elucidate whether NADPH- or NADH-oxidase functions are target proteins for MF interaction, the flavoprotein inhibitor diphenyleneiodonium chloride (DPI) was used. MF-induced free radical production was not inhibited by DPI, whereas tetradecanoylphorbolacetate (TPA)-induced free radical production was diminished by about 70%. TPA is known to induce a direct activation of NADPH-oxidase through the PKC pathway. Since DPI lacks an inhibitory effect in MF-exposed MBM cells, we suggest that 50 Hz MF stimulates the NADH-oxidase pathway to produce superoxide anion radicals, but not the NADPH pathway. Furthermore, we showed an oscillation (1-10 days) in superoxide anion radical release in mouse macrophages, indicating a cyclic pattern of NADH-oxidase activity.

Embryonal cardiotoxicity of the Helicobacter pylori lipopolysaccharide.

Helicobacter (H.) pylori is the causative agent of the peptic ulcer disease and a co-factor in the development of gastric malignancies. Recently, it has been maintained that chronic H. pylori infections in adults are linked to a higher risk of coronary heart diseases. In this respect, the acute toxic effects of the H. pylori lipopolysaccharide (LPS) on embryonal cardiomyocytes at different developmental stages was evaluated. White Leghorn chick embryos and smooth (S)--form NCTC 11637 strain H. pylori organisms were used. Both whole heath-killed H. pylori suspensions (3.10(6) bacteria/egg) and isolated S-LPS (500 ng/egg) or S-Lipid A (500 ng/egg) were non-lethal to 4-day embryos, becoming moderately lethal (5% to 30%) to 6- and 8-day embryos and highly lethal (> 90%) to 10- to 17-day embryos. The contractile activity of isolated atrial fragments from 10-day embryos was completely inhibited, within 5 min, following treatments with heath-killed H. pylori (3 x 10(6)/ml), or S-LPS (500 ng/ml), or S-Lipid A (500 ng/ml); the block determined by S-LPS and S-Lipid A was irreversible, while the block by bacterial suspensions was completely reversible upon withdrawal. Following a 24-hour treatment with S-LPS or S-Lipid A of single-cell cultures of cardiomyocytes (isolated from 10-day embryos) a dose-dependent cell loss was observed, as assessed by total protein dosage and direct counting of adherent cells. Propidium Iodide/Annexin V FACS-analysis confirmed the occurrence of cellular necrosis, but did not show any evidence of apoptotic processes. The release of superoxide anion radicals by cultured cardiomyocytes was as follows: S-Lipid A (25 micrograms/ml) > S-LPS (25 micrograms/ml) > heath killed H. pylori suspensions (3 x 10(6)/ml); control cultures did not release detectable amounts of superoxide anion radicals. Furthermore, cultured cardiomyocytes produced increased amounts of NO (N-monomethylarginine-inhibitable) following stimulation with S-LPS (25 micrograms/ml) or S-Lipid A (25 micrograms/ml) (but not heath killed H. pylori 3 x 10(6)/ml suspensions). Under all the above experimental conditions S-polysaccharide proved to be non-toxic. Concluding, H. pylori LPS is relatively non-toxic to the less differentiated cardiomyocytes; cardiomyocytes which are more advanced in their biochemical differentiation become highly sensitive to LPS and produce ROS and NO. ROS are probably responsible for the early toxic actions, while both ROS and NO are likely to be involved in the later degenerative/necrotic effects.

Bowman-Birk Inhibitor Suppresses Production of Superoxide Anion Radicals in Differentiated HL-60 Cells

The Bowman-Birk protease inhibitor (BBI) is a soybean-derived protease inhibitor with anticarcinogenic and anti-inflammatory properties. BBI has previously been shown to suppress the release of superoxide anion radicals from purified polymorphonuclear leukocytes. In the present study we evaluated the effect of BBI on the production of superoxide anion radicals in differentiated HL-60 cells. HL-60 cells are human lymphocytic cells that acquire neutrophil-like characteristics when treated with dimethyl sulfoxide or tetradecanoyl phorbol acetate. Superoxide anion radical production by differentiated HL-60 cells was measured in the presence of various concentrations of BBI or BBI concentrate, a soybean extract containing high levels of BBI. BBI was observed to suppress superoxide anion radical production by differentiated HL-60 cells in a dose-dependent manner. Extracts of differentiated HL-60 cells were also observed to produce superoxide anion radicals, but this activity was not affected by the presence of BBI. These results suggest that BBI inhibits superoxide anion radical generation in HL-60 cells but does not act as a simple free radical scavenger.

Ethanol metabolism by macrophages: possible role in organ damage.

Macrophages and hepatocytes oxidize ethanol to acetate in vitro at comparable rates but by different biochemical pathways. Ethanol metabolism by macrophages is largely ADH-independent and mainly based on cytochrome P450 and on the extracellular release of superoxide anion radicals. There is also evidence that during ethanol metabolism, macrophages release more acetaldehyde extracellularly than hepatocytes; the high concentrations of acetaldehyde around macrophages may damage surrounding tissue cells. Some of this acetaldehyde forms unstable cytotoxic complexes with serum albumin and with erythrocytes. The superoxide anion radicals released by macrophages may not only oxidize ethanol to acetaldehyde but also react with and damage cells in their immediate vicinity. After exposure to ethanol, macrophage-depleted rodents show markedly reduced levels of cytotoxic acetaldehyde-albumin complexes in the blood and reduced levels of hydroxyethyl radicals in the bile compared to control animals, indicating that the generation of such potentially pathogenic molecules is, to a large extent, dependent on macrophage activity. Macrophage-depleted animals also show less early liver damage than control animals. The reduction in ethanol-induced liver damage in macrophage-depleted mice and rats may be due to a reduction or elimination of the generation of various Kupffer-cell-derived hepatotoxic substances, including acetaldehyde and reactive oxygen radicals, in such animals. These data suggest that ethanol metabolism by tissue macrophages may play an important role in mediating ethanol-related tissue damage.

Review of alterations in endothelial nitric oxide production in diabetes: protective role of arginine on endothelial dysfunction.

Nitric oxide release from the endothelium plays an important role in regulation of vascular tone, inhibition of both platelet and leukocyte aggregation and adhesion, and inhibition of cell proliferation. These properties suggest that the level of NO production by the endothelium may play a pivotal role in the regulation of vascular disease. Analysis using mass spectrometry has revealed that NO is produced by NOS from the terminal guanidino nitrogen of the precursor amino acid L-arginine. Thus, utilization of L-arginine and conversion to NO may establish a regulatory site in the development of endothelial dysfunction. Endothelial dysfunction is characterized by defective endothelium-dependent relaxation, and some reviews regarding endothelial dysfunction in diabetes have been published. These reviews have focused on factors that might contribute to defective relaxation, some of which will not be addressed in detail in this review. The purpose of the present review is to summarize evidence that specifically supports either decreased NO production by diabetic vascular endothelium and/or impaired NO-mediated endothelium-dependent relaxation. Second, this review provides reasonable alternatives to explain some of the controversies in this research area. Third, since there is growing evidence that arginine appears to have some benefits for diabetes-associated abnormalities, this review summarizes the current state of knowledge of effects of acute and chronic administration of L-arginine on diabetesinduced endothelial dysfunction and discusses potential NOdependent and -independent mechanisms whereby therapeutic intervention with L-arginine might benefit the diabetic endothelium.

The human epidermis.

1993). Since undifferentiated keratinocytes (i.e. basal cells) provide the major supply of catecholamines to the total epidermis, it seems feasible that those basal cells in contact with melanocytes are of major importance in signal transduction, metabolism, growth and differentiation. Any local trauma to the epidermis by infection or physical damage can attract infiltrating lymphocytes to produce the biological ‘oxygen-burst’ leading to a local release of superoxide anion radicals

Cytotoxicity of dust constituents towards alveolar macrophages: Interactions of heavy metal compounds

The interactions between different heavy metal compounds which affect their cytotoxicity towards rabbit alveolar macrophages were investigated. The cells were exposed in vitro to combinations of As 3+, Cd 2+, Hg 2+, Ni 2+, or V 5+ with different concentrations of another heavy metal compound. Toxicity was determined as the depression of zymosan-induced release of superoxide anion radicals. Significant antagonisms occurred in the combinations Cd 2+/Zn 2+, Hg 2+/As 3+, and Hg 2+/Se 4+, while significant synergisms were exhibited by the combinations Cd 2+/Cu 2+, Cd 2+/Sn 2+, Hg 2+/Cu 2+, Ni 2+/Cd 2+, Ni 2+/Cu 2+, Ni 2+/Sn 2+ and V 5+/Cu 2+. In the combinations As 3+/Zn 2+, Hg 2+/Cd 2+ and Hg 2+/Zn 2+, both kinds of interactions were observed depending on the concentrations of the heavy metal compounds. An interpretation of the measured heavy metal interactions with reference to the toxicity of heavy metal-containing dusts is attempted.

Interaction of tocopherol with peroxyl radicals does not lead to the formation of lipid hydroperoxides in liposomes

α-Tocopherol (α-TOH) is thought to act as a lipid-soluble antioxidant by scavenging the chain-carrying lipid peroxyl radical (LOO .), giving rise to α-TO . and LOOH. The aim of the present work is to test the possibility that α-TOH may also exert a protective action by converting LOO . into the parent, unoxidized lipid molecule (LH) with concomitant formation of O 2 − and T +O. To assess the relative contribution of the two pathways, the amounts of products accumulating were measured, i.e. LOOH and O 2 −, under conditions where lipid peroxidation was initiated at linear rates by the UV light-induced decomposition of AIBN. The overall conclusion of the study is based on the observation that significant amounts of O 2 − were produced, whereas the ΔLOOH/Δα-TOH was considerably smaller than 1.0. A mechanism of one or two-electron oxidation of TOH is considered; according to it the phospholipid primary structure is restored (LOO . → LH, breaking of the CO bond). This leads to the oxygen or superoxide anion-radical release accordingly.

Cryptoporic acids A-G, drimane-type sesquiterpenoid ethers of isocitric acid from the fungus Cryptoporus volvatus

Seven novel bitter drimane-type sesquiterpenoids with an isocitric acid group named cryptoporic acids (CA) A-G have been isolated from the fungus Cryptoporous volvatus and their absolute stereostructures established by a combination of chemical transformation, NMR spectral data and X-ray crystallographic analysis. Cryptoporic acids strongly inhibited the release of superoxide anion radicals from guinea-pig peritoneal macrophage as well as rabbit polymorphonuclear leucocyte. CA-E showed anti-tumour promoting activity whilst CA-D slightly enhanced tumour promoting and protein kinase C activity. CAs also inhibited elongation of the second coleoptile and germination of rice in husk at 200 ppm.