Metabolism of ecdysteroids by a chitin-synthesizing insect cell line

A chitin-synthesizing cockroach cell line (UMBGE-4) previously shown to secrete ecdysteroids was analyzed for its ability to metabolize potential precursors of ecdysone (e.g., 2-deoxyecdysone, 2,22-dideoxyecdysone, 2,22,25-trideoxyecdysone, and cholesterol). All, except cholesterol, were actively metabolized by UMBGE-4 cells. However, all but 2-deoxyecdysone were converted to polar and hydrolyzable metabolites, and not to ecdysone. Labeling with cholesterol was unsuccessful. Labeling experiments with molting hormones, i.e., ecdysone and 20-hydroxyecdysone, confirmed that this cell line can metabolize ecdysteroids and allowed identification of some of the products. Molting hormones were converted into acetate conjugates and polar conjugates which were often double-conjugates, i.e., polar conjugates of acetate conjugates. Labeling experiments with ecdysone demonstrated that this cell line possesses a low ecdysone 20-hydroxylase activity. The capacity of UMBGE-2 cells, which do not synthesize chitin or ecdysteroids, was also examined. Neither ecdysone nor 20-hydroxyecdysone was significantly metabolized by UMBGE-2 cells. 2-Deoxyecdysone and 2,22-dideoxyecdysone were very slowly metabolized respectively to more polar compounds.     

Ecdysteroid hormones and metabolites of the stone crab, Menippe mercenaria

The Y-organs of the xanthid crab Menippe mercenaria secrete the ecdysteroids, 3-dehydroecdysone (3DE) and lesser amounts of 3-dehydro (or 2-dehydro)-25-deoxyecdysone (3D25dE) in vitro. These ecdysteroids were identified by elution-time comparisons with authentic standards, mass spectrography, and, for 3D25dE, infrared spectrometry. Tissues were incubated 18 hr with [(3)H]3DE. Activities representing 3beta-reductase and 20-hydroxylase generally were present, evidenced by finding in the tissue/medium extract labeled ecdysone (E) and 20-hydroxyecdysone (20E). Labeled 3-dehydro-20-hydroxyecdysone (3D20E) also appeared to be present. Tissue blanks and hemolymph were devoid of activity. Muscle was low, hypodermis was intermediate, and hindgut and gonads were high in activity of the enzymes. Consistent with the presence of these enzymes in peripheral tissues, ecdysteroid products identified in the hemolymph were 20E, 3D20E, and 25-deoxy-20-hydroxyecdysone (25d20E; ponasterone A). Structures of 20E and 3D20E were confirmed by co-elution with authentic standards in high-performance liquid chromatography (HPLC), co-elution of derivatives in gas chromatography, and mass spectroscopy. Ponasterone A (identified by HPLC co-elution with the standard), like 20E is present in the hemolymph in prominent amounts. These data indicate that Menippe, among crustaceans thus far studied, secretes a unique combination of ecdysteroid hormones, namely, a 3- (or 2-) oxo compound and a 25-deoxy compound. This represents a different kind of branch point from 5beta-diketol in ecdysteroid biosynthesis, in which the intermediate, 5beta-ketodiol is bypassed. A result is the joint appearance in the circulation of the hormones, 20E and ponasterone A, which in other species are singly prominent.     

Characterization of three hydroxylases involved in the final steps of biosynthesis of the steroid hormone ecdysone in Locusta migratoria (Insecta, Orthoptera)

It is most generally accepted that the last three enzymatic reactions in the biosynthetic pathway of ecdysone are, in this order, the hydroxylations at positions C-25, C-22 and C-2. Using high specific activity tritiated ecdysone precursors (2,22,25-trideoxyecdysone, 2,22-dideoxyecdysone and 2-deoxyecdysone) we have characterized the hydroxylases involved in these reactions, in the major biosynthetic tissue of ecdysone, i.e. the prothoracic glands. We show that C-2 hydroxylase is a mitochondrial oxygenase which differs from conventional cytochrome P-450-dependent monooxygenases by its relative insensitivity to CO. In contrast, C-22 and C-25 hydroxylases appear as classical cytochrome P-450 monooxygenases; C-22 hydroxylase is a mitochondrial enzyme whereas our data point to a microsomal localization of the C-25 hydroxylase.     

Phantom encodes the 25-hydroxylase of Drosophila melanogaster and Bombyx mori: a P450 enzyme critical in ecdysone biosynthesis

We have reported recently the identification and characterization of the last three mitochondrial cytochrome P450 enzymes (CYP) controlling the biosynthesis of 20-hydroxyecdysone, the molting hormone of insects. These are encoded by the following genes: disembodied (dib, Cyp302a1, the 22-hydroxylase); shadow (sad, Cyp315a1, the 2-hydroxylase); and shade (shd, Cyp314a1, the 20-hydroxylase). Employing similar gene identification and transfection techniques and subsequent biochemical analysis of the expressed enzymatic activity, we report the identity of the Drosophila gene phantom (phm), located at 17D1 of the X chromosome, as encoding the microsomal 25-hydroxylase (Cyp306a1). Similar analysis following differential display-based gene identification has also resulted in the characterization of the corresponding 25-hydroxylase gene in Bombyx mori. Confirmation of 2,22,25-trideoxyecdysone (3beta,5beta-ketodiol) conversion to 2,22-dideoxyecdysone (3beta,5beta-ketotriol) mediated by either Phm enzyme employed LC, MS and definitive NMR analysis. In situ developmental gene analysis, in addition to northern, western and RT-PCR techniques during Drosophila embryonic, larval and adult development, are consistent with this identification. That is, strong expression of phm is restricted to the prothoracic gland cells of the Drosophila larval ring gland, where it undergoes dramatic changes in expression, and in the adult ovary, but also in the embryonic epidermis. During the last larval-larval transition in Bombyx, a similar expression pattern in the prothoracic gland is observed, but as in Drosophila, slight expression is also present in other tissues, suggesting a possible additional role for the phantom enzyme.     

Further characterization of the 2-deoxyecdysone C-2 hydroxylase from Locusta migratoria

Additional data are provided on the enzyme 2-deoxyecdysone C-2 hydroxylase which has been shown in a previous study (Kappler et al., 1986) to be a mitochondrial hydroxylase with some classical characteristics of a cytochrome P-450 monooxygenase but which appeared to be insensitive to CO. Using ¹⁸O₂, we have now demonstrated that molecular oxygen is directly incorporated into ecdysone during the process of C-2 hydroxylation. Neither cumene hydroperoxide nor linoleyl hydroperoxide could support C-2 hydroxylation. When the reaction was sustained by α-ketoglutarate, addition of cofactors like Fe²⁺, ascorbate and catalase caused only a slight increase of the enzymatic activity whereas the α-ketoglutarate-dependent hydroxylation was largely decreased in the presence of malonate; these data eliminate the possible existence of a dioxygenase mechanism for C-2 hydroxylation.The paper also provides inhibition kinetics which indicate that 2-deoxy-20-hydroxyecdysone, 2,22-bisdeoxyecdysone and 2,22,25-trideoxyecdysone are competitive inhibitors of the C-2 hydroxylase whereas the 3-epi isomer of 2-deoxyecdysone is a non-competitive inhibitor.     

A possible role of 20-hydroxyecdysone in embryonic development of the silkworm Bombyx mori

It has been well established that eggs of insects, including those of the silkworm Bombyx mori, contain various ecdysteroids and the amounts of these ecdysteroids fluctuate during embryonic development. In order to know the function of egg ecdysteroids in embryonic development of B. mori, we examined the biological activities of various egg ecdysteroids by in vitro ligand-binding assay and bioassay using B. mori eggs. First, using the ecdysteroid receptor of B. mori (BmEcR-B1/BmUSP heterodimer) prepared by yeast and Escherichia coli expression systems, the interaction between the ecdysteroid receptor and various egg ecdysteroids of B. mori was analyzed. The relative binding affinities of egg ecdysteroids to the BmEcR-B1/BmUSP heterodimer decreased in the order of 20-hydroxyecdysone > 2-deoxy-20-hydroxyecdysone > 22-deoxy-20-hydroxyecdysone > ecdysone > 2-deoxyecdysone > ecdysone 22-phosphate. Next, several egg ecdysteroids of B. mori were injected into the prospective diapause eggs, which show a very low level of free ecdysteroids at the onset of embryonic diapause (gastrula stage). Approximately 7% of them (P < 0.002, chi(2)-test) developed beyond the gastrula stage without entering diapause by the injection of 20-hydroxyecdysone (25 ng/egg). In contrast, the injection of other ecdysteroids was not effective in inducing embryonic development. These results suggest that 20-hydroxyecdysone, via the ecdysteroid receptor, is responsible for the developmental difference between diapause and non-diapause in B. mori embryos. Furthermore, it was suggested that continuous supply of 20-hydroxyecdysone may be required to induce embryonic development.     

Ovarian ecdysteroids and their secretion in late-pharate adults of Galleria mellonella

Approximately two-thirds of the total amount of ecdysteroids in late—pharate adults of the wax moth, Galleria mellonella, were found in the ovaries and one-third in the ovariectomized body. Chemical analysis of these ecdysteroids by thin-layer and high-pressure liquid chromatography, coupled with an ecdysteroid radioimmunoassay, revealed the presence of 2-deoxyecdysone, ecdysone and 20-hydroxyecdysone, as well as high and low polarity unknowns. The predominant identifiable ecdysteroid in both the ovaries and ovariectomized body was 2-deoxyecdysone, followed by lesser amounts of ecdysone and 20-hydroxyecdysone, respectively. Incubation of late-pharate adult ovaries in culture medium revealed that they synthesize and secrete ecdysteroids in vitro. The in vitro distribution of ecdysteroids between ovaries and incubation medium was similar to that observed between ovaries and ovariectomized bodies in situ and the predominant identifiable moiety both retained and released by the ovaries in vitro was 2-deoxyecdysone, followed by lesser amounts of ecdysone and 20-hydroxyecdysone. Collectively, these results support the idea that the ecdysteroids synthesized by the ovaries of late-pharate adult Galleria are both stored and secreted and that the quantity of a specifically secreted ecdysteroid is precisely controlled. This apparent regulation of the distribution of ovarian ecdysteroids raises the possibility that the stored and secreted forms have distinct functions in the reproductive physiology of this insect.     

The identification and titres of conjugated and free ecdysteroids in developing ovaries and newly-laid eggs of Schistocerca gregaria

Ecdysteroid levels throughout ovarian development and in newly-laid eggs of S. gregaria have been determined. A simple method for the separation of free and conjugated ecdysteroids is described. Both free and polar conjugated ecdysteroids are present at the end of oögenesis and in newly-laid eggs, but the polar conjugated ecdysteroids always predominate; 95% of the total ecdysteroid in newly-laid eggs is in the conjugated form. Ecdysone, 2-deoxyecdysone and 20-hydroxyecdysone have been fully characterized from both the ‘free’ and ‘conjugated’ fractions. The presence of traces of 26-hydroxyecdysone in the ‘conjugate’ fraction was indicated by HPLC analyses. The levels of ecdysteroid released from the conjugates of newly-laid eggs were 35 μg/egg pod (44 μg/g wet weight) for ecdysone, 16 μg/egg pod (19.4 μg/g) for 2-deoxyecdysone and 5 μg/egg pod (6.1 μg/g) for 20-hydroxyecdysone. The level of free ecdysone found in newly-laid eggs was 2 μg/egg pod (2.6 μg/g).     

The effect of glucose on the activity of phosphofructokinase in the mucosa of rat small intestine.

Maturing eggs of the desert locust, Schistocerca gregaria, contain a variety of ecdysteroid (insect moulting hormone) conjugates and metabolites, four of which have been previously isolated from polar extracts and identified as ecdysonoic acid, 20-hydroxyecdysonoic acid, 3-acetylecdysone 2-phosphate and ecdysone 2-phosphate. In the present study we have isolated eight additional ecdysteroids from similar late-stage eggs by high-performance liquid chromatography. The 22-phosphate esters of ecdysone, 2-deoxyecdysone, 20-hydroxyecdysone and 2-deoxy-20-hydroxyecdysone, all of which were first identified as ecdysteroid components of newly-laid eggs of S. gregaria, were identified by co-chromatography with authentic compounds and by physicochemical techniques. The remaining compounds were identified as 3-acetyl-20-hydroxyecdysone 2-phosphate, 3-epi-2-deoxyecdysone 3-phosphate, 3-acetylecdysone 22-phosphate and 2-acetylecdysone 22-phosphate by fast atom bombardment mass spectrometry, p.m.r. spectroscopy and analysis of the steroid moieties after enzymic hydrolysis. The latter two compounds, after isolation, are susceptible to nonenzymic acetyl migration and deacetylation to give mixtures of ecdysone 22-phosphate and its 2- and 3-acetate derivatives. The possible role and significance of these ecdysteroid conjugates with respect to the control of hormone titres in insect eggs is discussed.     

The first cytochrome P450 in ferns. Evidence for its involvement in phytoecdysteroid biosynthesis in Polypodium vulgare

The fern Polypodium vulgare is a phytoecdysteroid (PE)-producing plant. Cultures of P. vulgare prothalus produce PE, whereas prothalus-derived callus cultures do not. However, this callus line can transform topically applied ecdysone (E) to 20-hydroxyecdysone (20E), which is the last step in the biosynthetic pathway of the main plant PE. This hydroxylation is catalysed by a cytochrome P450 enzyme. E treatment of the callus line results in an increased amount of P450, showing a linear correspondence between the amount of P450 and in vivo E 20-hydroxylation activity, estimated by measuring the bioconversion of E to 20E. This activity can be inhibited by molecules that bind to the P450-heme group. E shows a P450-substrate-binding spectrum with microsomes that overexpress the P450 protein. Finally, a P450 protein was purified from E-treated calli, this being the first P450 to be described in the pterydophyte group.     

Entomogenous fungus Nomuraea rileyi inhibits host insect molting by C22-oxidizing inactivation of hemolymph ecdysteroids

The entomogenous fungus Nomuraea rileyi reportedly secretes a proteinaceous substance inhibiting larval molt and metamorphosis in the silkworm Bombyx mori. We studied the possibility that N. rileyi controls B. mori development by inactivating hemolymph molting hormone, ecdysteroids. Incubation of ecdysone (E) and 20-hydroxyecdysone (20E) in fungal-conditioned medium resulted in their rapid modification into products with longer retention times in reverse-phase HPLC. Each modified product from E and 20E was purified by HPLC, and identified by NMR as 22-dehydroecdysone and 22-dehydro-20-hydroxyecdysone. Some other ecdysteroids with a hydroxyl group at position C22 were also modified. Injection of the fungal-conditioned medium into Bombyx mori larvae in the mid-4th instar inhibited larval molt but induced precocious pupal metamorphosis, and its injection into 5th instar larvae just after gut purge blocked pupal metamorphosis. In hemolymph of injected larvae, E and 20E disappeared and, in turn, 22-dehydroecdysone and 22-dehydro-20-hydroxyecdysone accumulated. These results indicate that N. rileyi secretes a specific enzyme that oxidizes the hydroxyl group at position C22 of hemolymph ecdysteroids and prevents molting in B. mori larvae.     

Developmental Changes of Ecdysteroids in the Eggs of the Silkworm, Bombyx mori

Ecdysteroids were studied in relation to embryonic development and diapause of the silkworm, Bombyx mori . The majority of the ecdysteroids was found to be in the conjugated form, and minor part, in the free form. In the developing eggs, 2-deoxyecdysone, 2-deoxy-20-hydroxy-ecdysone and 3-epi-ecdysone were found to have the free ecdysteroid form as well as the conjugated ecdysteroid form. Ecdysone and 20-hydroxyecdysone almost exclusively had the conjugated form. The concentration of ecdysteroids in the embryo was higher than that in yolkplasm in the early embryonic stages. During the embryonic diapause, the concentration of free ecdysteroids decreased to a low level while the conjugated form maintained the original level.     

Identification of the 22-phosphate esters of ecdysone, 2-deoxyecdysone, 20-hydroxyecdysone and 2-deoxy-20-hydroxyecdysone from newly laid eggs of the desert locust, Schistocerca gregaria.

The four major ecdysteroid (insect moulting hormone) conjugates present in the newly laid eggs of the desert locust, Schistocera gregaria, have been purified by reversed-phase and anion-exchange high-performance liquid chromatography. The steroid moieties were identified as ecdysone, 2-deoxyecdysone, 20-hydroxyecdysone and 2-deoxy-20-hydroxyecdysone. Phosphate analysis of acid-hydrolysed samples showed a steroid:phosphate ratio of approx. 1:1 for all four compounds. The intact conjugates were identified as ecdysone 22-phosphate, 2-deoxyecdysone 22-phosphate, 20-hydroxyecdysone 22-phosphate and 2-deoxy-20-hydroxyecdysone 22-phosphate by fast atom bombardment mass spectrometry and 1H, 13C and 31P n.m.r. The significance of ecdysteroid phosphates as a source of free hormone during embryogenesis is discussed.     

Isolation and identification of major ecdysteroids from the pycnogonidPycnogonum litorale (Ström) (Arthropoda,Pantopoda)

Summary From adults ofPycnogonum litorale (Ström) eight ecdysteroids were isolated by HPLC and identified by mass spectrometry and NMR. One of the compounds is 20-hydroxyecdysone, two further ecdysteroids show no OH-group at C-22 (22-deoxy-20,26-dihydroxyecdysone, 22-deoxy-20-hydroxyecdysone=taxisterone). The five other compounds are esters of ecdysteroids with acetic acid (25R and 25S isomers of 20,26-dihydroxyecdysone 22-acetate, 20-hydroxyecdysone 22-acetate) or with glycolic acid (20-hydroxyecdysone 22-glycolate, ecydsone 22-glycolate). The latter are new among zoo- and phytoecdysteroids. No significant amounts of ecdysone could be detected. The origin of the ecdysteroids inPycnogonum litorale and their biological activity are discussed.Abbreviations RP-HPLC Reversed-phase high performance liquid chromatography - NP normal phase - RIA radioimmunoassay - NMR nuclear magnetic resonance - FT Fourier transform - CI/D chemical ionization/desorption - TFA trifluoroacetic acid - E ecdysone - 20E 20-hydroxyecdysone - 2026E 20 26-dihydroxyecdysone     

Ecdysteroids in Carausius eggs during embryonic development

Peaks of ecdysteroids were observed during the different phases of embryonic development of intact Carausius eggs or eggs precociously deprived of their exochorion and cultivated under paraffin oil. Several groups of ecdysteroids were separated and analyzed by thin-layer chromatography (TLC) and high performance liquid chromatography (HPLC) combined with radioimmunoassay. Ecdysteroids were similar in the two categories of eggs, including high-polarity products (essentially conjugates hydrolyzable by Helix pomatia digestive juice, or alkaline phosphatase), possible ecdysonoic acids (unhydrolyzable polar substances), free hormones, and nonpolar ecdysteroids. Four ecdysteroids were identified by co-elution during HPLC with reference compounds of 20,26-dihydroxyecdysone, 20-hydroxyecdysone, ecdysone, and 2-deoxy-20-hydroxyecdysone. Concentrations of these substances (free and conjugated forms) were studied during the different stages of embryonic development: 20-hydroxyecdysone and 2-deoxy-20-hydroxyecdysone were the major free ecdysteroids. They showed parallel variations with large peaks at stages VI₈ and VII₆ whereas ecdysone titers were consistently low. Injected labelled ecdysone was converted efficiently into 20-hydroxyecdysone, and both compounds underwent 26-hydroxylation and/or conjugation to polar or apolar metabolites.     

Effects of ecdysteroids on Chlorella vulgaris

The effects of three ecdysteroids, 20-hydroxyecdysone (20E), 2-deoxy-20-hydroxyecdysone (2d20E) and 20-hydroxyecdysone 22-acetate (20E22Ac), on growth and the levels of cellular components in Chlorella vulgaris Beijerinck (Trebouxiophyceae) are reported and compared with data previously reported for ecdysone (E; Bajguz A and Koronka A, Plant Physiol Biochem 39: 707–715, 2001). All three 20-hydroxyecdysteroids stimulate growth of C. vulgaris cells over a wide concentration range (10⁻¹⁶ to 10⁻⁷  M ). Optimal stimulation is observed at 10⁻⁹  M with each ecdysteroid. High concentrations (>10⁻⁶  M ) are cytotoxic. The potency ranking of the ecdysteroids is 20E > 20E22Ac > 2d20E > E. Levels per cell of DNA, RNA, protein, sugars, organic and inorganic phosphorus, chlorophylls a and b and phaeophytins a and b are all stimulated by ecdysteroid treatment when compared with the untreated control cells. Possible modes of action of ecdysteroids on C. vulgaris cells are discussed.     

Conversion of ecdysone and 20-Hydroxyecdysone into 26-OIC derivatives is a major pathway in larvae and pupae of species from three insect orders

Insects convert ecdysone and 20-hydroxyecdysone into their corresponding 26-oic derivatives, named ecdysonoic acid and 20-hydroxyecdysonoic acid respectively. The conversion takes piace in several tissues and can either be the only pathway for converting ecdysone into highly polar ecdysteroids, or coexist with various conjugating mechanisms. 20-Hydroxyecdysonoic acid was isolated from Pieris brassicae pupae as its methyl ester derivative. Its chemical structure was identified by Cl/D mass spectrometry and compared with a synthetic compound (20-hydroxy-25-deoxyecdysonoic acid) chemically prepared by oxidation of inokosterone (20,26-dihydroxy-25-deoxyecdysone). Natural ecdysonoic acids appear to exist as a mixture of 25R and 25S isomers. The significance of this pathway is discussed in comparison with similar reactions occuring in the metabolism of steroid hormones in vertebrates.     

Mechanism of formation of the A/B cis ring junction of ecdysteroids in Polypodium vulgare.

1. The fates of the alpha-, 4 alpha- and 4 beta-hydrogen atoms of cholesterol during formation of the A/B cis ring junction of ecdysteroids was investigated by administration of [4-14C, 3 alpha-3H], [4-14C, 4 alpha-3H]- and [4-14C, 4 beta-3H]cholesterol species to the fern, Polypodium vulgare, and isolation of the 20-hydroxyecdysone formed in each case. 2. The 3H was retained in the ecdysteroid formed from each substrate. 3. Location of the 3H in the 20-hydroxyecdysone indicated that migration of 3H from the 3 alpha- and 4 beta-positions to C-4 and C-5, respectively, had occurred, whereas the 4 alpha-3H atom was retained at C-4. 4. A possible mechanism for the formation of the A/B cis ring junction of ecdysteroids in P. vulgare is presented.     

Metabolism of ecdysteroid by testes of the tobacco budworm,Heliothis virescens

Testes from late last stage larvae of the tobacco budworm, Heliothis virescens, were incubated with [³H]ecdysone and [³H]cholesterol. [³H]Ecdysone was converted to six other major ecdysteroids, identified by cochromatography in reverse-phase high-pressure liquid chromatography (RPHPLC); four of them were verified by normal-phase HPLC. A highly polar fraction, moderately polar ecdysteroids (20,26-dihydroxyecdysone, 3-epi-20-hydroxyecdysone, and 20-hydroxyecdysone) and low-polarity ecdysteroids, including 2-deoxyecdysone, were detected after incubation with [³H]ecdysone. Compounds that reacted positively to antibodies to progesterone and testosterone were detected in the low-polarity fractions. Testes were incubated in fractions corresponding to each of the major ecdysteroid peaks derived from [³H]ecdysone metabolism. Although most of the radioactive ecdysteroid fractions were further metabolized to high- and low-polarity endpoints, 88% of the [³H]20-hydroxyecdysone peak apparently remained unmetabolized. 20-Hydroxyecdysone may be the primary ecdysteroid product of testes of H. virescens. [³H]Cholesterol was not metabolized to any appreciable extent.     

Antioxidative and free radical scavenging effects of ecdysteroids from Serratula strangulata

The antioxidative and free radical scavenging effects of four ecdysteroids, 20-hydroxyecdysone (E1), 25-deoxy-11,20-dihydroxyecdysone (E2), 24-(2-hydroxyethyl)-20-hydroxyecdysone (E3), and 20-hydroxyecdysone-20,22-monoacetonide (E4), isolated from the Chinese herb Serratula strangulata have been investigated in vitro. These ecdysteroids could protect human erythrocytes against oxidative hemolysis induced by a water-soluble azo initiator 2,2'-azobis(2-amidinopropane hydrochloride) (AAPH). They could also inhibit the peroxidation of rat liver microsomes induced by hydroxyl radicals, as monitored by the formation of thiobarbituric acid reactive substances (TBARS), and prevent radical-induced decrease of membrane fluidity as determined by fluorescence polarization. They reacted with galvinoxyl radicals in homogeneous solution, and the pseudo-first-order rate constants were determined spectrophotometrically by following the disappearance of galvinoxyl radicals. Compounds E1 and (or) E3 were the most active in both antioxidative and radical-scavenging reactions.