Carotenoids as cellular antioxidants.

Consumption of carotenoids is associated with an enhanced immune response and protection against neoplasia and atherosclerosis. Because these effects have been achieved using carotenoids with no pro-vitamin A activity, they are assumed to be due to the antioxidant properties of carotenoids. Carotenoids protect against photosensitized oxidation by quenching singlet oxygen. In addition, beta-carotene reacts chemically with peroxyl radicals to produce epoxide and apocarotenal products. To investigate the potential significance of these reactions to biological systems, we have used soybean lipoxygenase to generate peroxyl radical enzymatically. beta-Carotene inhibits the oxidation of linoleic acid by soybean lipoxygenase as well as the formation of the hydroperoxide product. In addition, the absorption of beta-carotene is diminished (bleached) by soybean lipoxygenase. The potential significance of these antioxidant reactions of carotenoids to biological function is discussed.     

Chemical studies on antioxidant mechanism of tea catechins: analysis of radical reaction products of catechin and epicatechin with 2,2-diphenyl-1-picrylhydrazyl

Tea catechins, an important class of polyphenols, have been shown to have wide spectrum of antitumor activity believed to be due mainly to their antioxidative effect. In this study, the radical scavenging behavior of catechins on 2,2-diphenyl-1-picrylhydrazyl (DPPH) was studied. Two reaction products of (+)-catechin, and two reaction products of (-)-epicatechin were purified and identified. Their structures were determined on the basis of detailed high-field 1-D and 2-D NMR spectral analysis. Structure elucidation of these products can provide insights into specific mechanisms of antioxidant reactions. A possible mechanism of the formation of reaction products is suggested.     

Antioxidant mechanisms of isoflavones in lipid systems: paradoxical effects of peroxyl radical scavenging

Oxidation of lipids has been implicated in the pathophysiology of atherosclerosis. It has been suggested that scavenging of lipid peroxyl radicals contribute to the antiatherosclerotic effects of naturally occurring compounds such as the isoflavones. This group of polyphenolics includes genistein and is present in relatively high concentrations in food products containing soy. Soy isoflavones are capable of inhibiting lipoprotein oxidation in vitro and suppressing formation of plasma lipid oxidation products in vivo. However, key aspects of the antioxidant mechanisms remain unknown. In this study the antioxidant effects of genistein and other soy isoflavones on lipid peroxidation initiated by mechanistically diverse oxidants was investigated. Although isoflavones inhibited lipid peroxidation stimulated by both metal-dependent and independent processes, the concentration required for these effects were relatively high compared to those found in vivo. Interestingly, however, isoflavones were not consumed and remained in the native state over the time during which inhibition of lipid peroxidation was observed. This was also the case under conditions where synergistic inhibition of LDL oxidation was observed with ascorbate. Furthermore, in an oxidation system driven solely by peroxyl radicals, isoflavones were found to be relatively poor peroxyl radical scavengers. Consistent with the apparent lack of reactivity with lipid-derived oxidants, isoflavones were also relatively resistant to oxidation mediated by the potent oxidant peroxynitrite. The potential antioxidant mechanisms of isoflavones are discussed in the context of possible reactivities of isoflavone-derived phenoxyl radicals.     

Nitroxidation, nitration, and oxidation of a BODIPY fluorophore by RNOS and ROS.

BODIPY C11 581/591 (BODIPY11) represents a sensitive probe for quantification of relative antioxidant capacity. However, the mechanism of BODIPY11 fluorescence decay in the presence of reactive oxygen species (ROS) and reactive nitrogen oxide species (RNOS) requires clarification. Azo-initiators provide a continuous source of peroxyl radicals that in simple, aerobic, homogeneous, buffered solution simulate lipid peroxyl radical formation. Inhibition of BODIPY11 fluorescence decay was assayed and quantified for several families of antioxidants, including phenols, NO donors, and thiols. Fluorescence decay of BODIPY11 in these systems demonstrated similar patterns of antioxidant activity to those observed in classical oxygen pressure measurements, and provided a readily applied quantification of antioxidant capacity and mechanistic information, which was analyzed by measurement of induction periods, initial rates, and net oxidation. LC/MS analysis confirmed that peroxyl radical-induced irreversible fluorescence decay of the BODIPY11 fluorophore is due to oxidative cleavage of the activated phenyldiene side chain. The behavior of BODIPY11 towards RNOS was more complex, even in these simple systems. Incubation of BODIPY11 with bolus peroxynitrite or a sydnonimine peroxynitrite source produced a variety of novel products, characterized by LC/MS, derived from oxidative cleavage, nitroxidation, and nitration reactions. The "NO scavenger" PTIO reinforced the antioxidant activity of NO, and inhibited BODIPY11 oxidation induced by the sydnonimine. These observations suggest that BODIPY11 is a well-behaved fluorescence probe for peroxidation and antioxidant studies, but that for study of RNOS even co-application of fluorescence decay with LC/MS measurements requires careful analysis and interpretation.     

Antioxidant activity of carotenoids: An electron-spin resonance study on β-carotene and lutein interaction with free radicals generated in a chemical system

β-Carotene is thought to be a chain-breaking antioxidant, even though we have no information about the mechanism of its antioxidant activity. Using electron-spin resonance (ESR) spectroscopy coupled to the spin-trapping technique, we have studied the effect of β-carotene and lutein on the radical adducts of the spin-trap PBN (N-t -butyl-α-phenylnitrone) generated by the metal-ion breakdown of different tert -butyl hydroperoxide (t BOOH) concentrations in methylene chloride. The peroxyl radical, along with an oxidation product of PBN (the PBNOx), trapped at room temperature from the breakdown of high concentration of t BOOH (1 M), were quenched by β-carotene or lutein, in competition with the spin-trapping agent. However, carotenoids were not able to quench the alkoxyl and methyl radicals generated in the reaction carried out in the presence of low t BOOH concentration (1 mM). The reaction between carotenoids and the peroxyl radical was also carried out in the absence of the spin trap, at 77 K: Under these different experimental conditions, we did not detect any radical species deriving from carotenoids. In the same system, a further evidence of the peroxyl radical quenching by β-carotene and lutein was obtained. The antioxidant activity of vitamin E was also tested, for comparison with the carotenoids. In the presence of α-tocopherol, peroxyl and alkoxyl radicals were quenched, and the tocopheroxyl radical was detected. Our data provide the first direct evidence that carotenoids quench peroxyl radicals. Under our experimental conditions, we did not detect any carotenoid radical species that could derive from the interaction with the peroxyl radical. The radical-trapping activity of β-carotene and lutein demonstrated in this chemical reaction contributes to our understanding carotenoid antioxidant action in biological systems. © 1998 John Wiley & Sons, Inc. J Biochem Toxicol 12: 299–304, 1998     

Co-oxidation of xenobiotics: lipid peroxyl derivatives as mediators of metabolism

Systems which carry out peroxyl-dependent oxidations can serve as activation systems for carcinogenic compounds. Some function via classical peroxidase reactions in which an enzyme-derived oxidant performs the electron abstraction from or oxygen donation to the oxidizable substrate. This mechanism applies to the peroxidative activation of aromatic amines and of the phenolic compound diethylstilbestrol. These classical peroxidase reactions may be initiated by hydrogen peroxide or by organic peroxides, including lipid hydroperoxides. A different mechanism is involved in the oxygenation of polycyclic aromatic hydrocarbons and of aflatoxin B1. In these cases the oxidant is a peroxyl radical, and the reaction occurs by the direct, non-enzymatic interaction of the peroxyl radical and the oxidizable substrate. Most peroxyl radicals in biological systems are lipid-derived. The key reaction which distinguishes the peroxyl radical-dependent oxidations from the classical peroxidase reactions is the ability of the former to epoxidize activated carbon-carbon double bonds. The epoxidation of benzo[a]pyrene derivatives has been studied extensively in subcellular and whole cell and tissue systems, and is discussed as a model for this class of reaction. Determining the generality of this activation path and its role in vivo present the major questions to be answered in regard to the importance of these reactions in chemical carcinogenesis.     

Bile acids: antioxidants or enhancers of peroxidation depending on lipid concentration

Using three different assay systems, we have discovered a heretofore unrecognized antioxidant property of bile acids at physiological concentrations. Bile acids inhibit peroxidation of the polyunsaturated lipid, linoleic acid, and of the highly fluorescent protein phycoerythrin. In part, the antioxidant activity results from scavenging of peroxyl radicals by direct oxidation of the bile acids. The most abundant products of the reaction of cholate and chenodeoxycholate with peroxyl radicals were studied in detail and shown to be the keto derivatives formed by oxidation of the 7 alpha-hydroxyl groups. Paradoxically, at linoleate concentrations higher than 1-2 mM, glycocholate up to approximately 10-14 mM enhances lipid peroxidation and inhibits only at higher concentrations. These findings may prove important in understanding the etiology of certain disease states of the biliary tract and intestine where lipid peroxidation may be involved and in providing a rationale for the positive epidemiological correlation between high lipid intake and higher fecal bile acid output and colon cancer.     

Numerical revelation of the kinetic significance of individual steps in the reaction mechanism of methyl linoleate peroxidation inhibited by alpha-tocopherol

A kinetic model was constructed to describe the reactions involved in the oxidation of methyl linoleate (ML) inhibited by alpha-tocopherol (TH). The initial model of the reaction mechanism included 53 individual steps, which were numerically analyzed by the value method based on Hamiltonian systematization of kinetic equations. Good accord was obtained with experimental data at 40 and 50 degrees C. The dominant steps responsible for the antioxidant and pro-oxidant properties of TH in the process of ML peroxidation were revealed. Tocopherol-mediated peroxidation (TMP) and generation of alkoxyl radicals as a result of the reduction of hydroperoxides by TH or the decomposition tocopherol alkyl peroxides are the dominant reactions responsible for the pro-oxidant activities of alpha-tocopherol. The extreme behavior of reaction induction period in relation to TH initial concentration is related to the increase in the ratios of [tocopheroxyl radical]/[peroxyl radical] and the TMP rate/rate of termination by combination of tocopheroxyl and peroxyl radicals.     

Metal chelators react also with reactive oxygen and nitrogen species

Popular chelators (desferrioxamine, SIH, EDTA, EGTA, DTPA, and NTA) were demonstrated to have antioxidant properties, being able to reduce ABTS radical cation and react with peroxyl radicals, peroxynitrite, and hypochlorite. Desferrioxamine and SIH were most potent antioxidants in all cases. These results point to the necessity of a careful interpretation of experiments in which the inhibition of free radical reactions by antioxidants is used as a proof of involvement of metal ions in a reaction.     

Characterization of the oxidation products of BO-653 formed during peroxyl radical-mediated oxidation of human plasma

4,6-Di-tert-butyl-2,3-dihydro-2,2-dipentyl-5-benzofuranol (BO-653) is a novel antioxidant synthesized by theoretical findings and considerations. Here we report on the aqueous peroxyl radical-induced oxidation of human plasma in the presence of BO-653. When BO-653 was given to healthy human subjects at 400 mg twice daily for 28 days, lipids in the resulting plasma were protected from oxidation compared with lipids present in plasma from subjects receiving placebo. Similarly, BO-653 added in vitro at 50 muM inhibited the peroxyl radical-induced accumulation of cholesteryl ester hydroperoxides that occurred in the presence of alpha-tocopherol, although BO-653 did not decrease the rate of consumption of ascorbate, albumin-bound bilirubin, and uric acid. The antioxidant action of in vivo and in vitro added BO-653 was associated with the formation of two major reaction products of BO-653, the structures of which were elucidated by mass spectrometry and nuclear magnetic resonance analyses. The products were identified as stereoisomers of dioxybis(4,6-di-tert.-butyl-2,3,5,7a-tetrahydro-2,2-dipentylbenzofuran-5-one). These dialkylperoxides of BO-653 might be useful markers to assess the antioxidant function of BO-653 in biological systems in vivo.     

Nitric oxide reaction with lipid peroxyl radicals spares alpha-tocopherol during lipid peroxidation. Greater oxidant protection from the pair nitric oxide/alpha-tocopherol than alpha-tocopherol/ascorbate

The reactions of nitric oxide ((.)NO) and alpha-tocopherol (alpha-TH) during membrane lipid oxidation were examined and compared with the pair alpha-TH/ascorbate. Nitric oxide serves as a more potent inhibitor of lipid peroxidation propagation reactions than alpha-TH and protects alpha-TH from oxidation. Mass spectrometry, oxygen and (.)NO consumption, conjugated diene analyses, and alpha-TH fluorescence determinations all demonstrated that (.)NO preferentially reacts with lipid radical species, with alpha-TH consumption not occurring until (.)NO concentrations fell below a critical level. In addition, alpha-TH and (.)NO cooperatively inhibit lipid peroxidation, exhibiting greater antioxidant capacity than the pair alpha-TH/ascorbate. Pulse radiolysis analysis showed no direct reaction between (.)NO and alpha-tocopheroxyl radical (alpha-T(.)), inferring that peroxyl radical termination reactions are the principal lipid-protective mechanism mediated by (.)NO. These observations support the concept that (.)NO is a potent chain breaking antioxidant toward peroxidizing lipids, due to facile radical-radical termination reactions with lipid radical species, thus preventing alpha-TH loss. The reduction of alpha-T(.) by ascorbate was a comparatively less efficient mechanism for preserving alpha-TH than (.)NO-mediated termination of peroxyl radicals, due to slower reaction kinetics and limited transfer of reducing equivalents from the aqueous phase. Thus, the high lipid/water partition coefficient of (.)NO, its capacity to diffuse and concentrate in lipophilic milieu, and a potent reactivity toward lipid radical species reveal how (.)NO can play a critical role in regulating membrane and lipoprotein lipid oxidation reactions.     

Synergistic interaction between the probucol phenoxyl radical and ascorbic acid in inhibiting the oxidation of low density lipoprotein.

Chain-breaking antioxidants such as butylated hydroxytoluene, alpha-tocopherol, and probucol have been shown to decrease markedly the oxidative modification of low density lipoprotein (LDL). Their mechanism of action appears to involve scavenging of LDL-lipid peroxyl radicals. The purpose of this study was to investigate the occurrence of radical reactions produced during oxidation of LDL and LDL-containing probucol initiated by lipoxygenase or copper. In addition, we have investigated the possibility of a synergistic interaction between ascorbate and probucol in inhibiting the oxidation of LDL. Incubation of LDL-containing probucol and lipoxygenase produced a composite electron spin resonance (ESR) spectrum due to the endogenous alpha-tocopheroxyl radical and probucol-derived phenoxyl radical. The spectral assignment was further verified by chemical oxidation of alpha-tocopherol and probucol. In the presence of ascorbic acid, these radicals in the LDL particle were reduced to their parent compounds with concomitant formation of the ascorbate radical. In both the peroxidation of linoleic acid and the copper-initiated peroxidation of LDL, the antioxidant activity of probucol was significantly increased by low (3-6 microM) concentrations of ascorbate. The probucol-dependent inhibition of LDL oxidation was enhanced in the presence of ascorbic acid. We conclude that the reaction between the phenoxyl radical of probucol and ascorbate results in a synergistic enhancement of the antioxidant capacity of these two compounds and speculate that such reactions could play a role in maintaining the antioxidant status of LDL during oxidative stress in vivo.     

Lipid peroxyl radical intermediates in the peroxidation of polyunsaturated fatty acids by lipoxygenase. Direct electron spin resonance investigations

Lipid peroxyl radicals resulting from the peroxidation of polyunsaturated fatty acids by soybean lipoxygenase were directly detected by the method of rapid mixing, continuous-flow electron spin resonance spectroscopy. When air-saturated borate buffer (pH 9.0) containing linoleic acid or arachidonate acid was mixed with lipoxygenase, fatty acid-derived peroxyl free radicals were readily detected; these radicals have a characteristic g-value of 2.014. An organic free radical (g = 2.004) was also detected; this may be the carbon-centered fatty acid free radical that is the precursor of the peroxyl free radical. The ESR spectrum of this species was not resolved, so the identification of this free radical was not possible. Fatty acids without at least two double bonds (e.g. stearic acid and oleic acid) did not give the corresponding peroxyl free radicals, suggesting that the formation of bisallylic carbon-centered radicals precedes peroxyl radical formation. The 3.8-G doublet feature of the fatty acid peroxyl spectrum was proven (by selective deuteration) to be a hyperfine coupling due to a gamma-hydrogen that originated as a vinylic hydrogen of arachidonate. Arachidonate peroxyl radical formation was shown to be dependent on the substrate, active lipoxygenase, and molecular oxygen. Antioxidants are known to protect polyunsaturated fatty acids from peroxidation by scavenging peroxyl radicals and thus breaking the free radical chain reaction. Therefore, the peroxyl signal intensity from micellar arachidonate solutions was monitored as a function of the antioxidant concentration. The reaction of the peroxyl free radical with Trolox C was shown to be 10 times slower than that with vitamin E. The vitamin E and Trolox C phenoxyl radicals that resulted from scavenging the peroxyl radical were also detected.     

Comparative free radical chemistry in the radiolytic deamination and dephosphorylation of bio-organic molecules. II. Oxygenated solutions

The radiolytic deamination and dephosphorylation of peptides and phosphate esters in oxygenated solutions are OH-induced and involve the formation and subsequent degradation of the peroxyl radicals RCONHC(O2)R2 and POC(O2)R2 respectively. Reaction analogues in the degradation chemistry of these peroxyl intermediates from various peptides and phosphate esters including protein and DNA are evaluated in detail. The evidence is that the reaction mechanisms recently proposed for removal of POC(O2)R2 radicals in the radiolytic scission of the DNA strand are identical in form with the mechanisms identified in earlier studies of RCONHC(O2)R2 radicals in the radiolytic cleavage of the peptide chain. The analogy includes the chemical forms of the final products and their specific yields.     

Inhibition of radiation-induced lipid peroxidation by tetrahydrocurcumin: possible mechanisms by pulse radiolysis

The antioxidant property of tetrahydrocurcumin (THC), a reduced derivative of curcumin, was examined by its ability to inhibit radiation-induced lipid peroxidation in rat liver microsomes and compared with curcumin. The lipid peroxidation caused by irradiation of N2O-purged and aerated buffered aqueous solutions was found to be inhibited by THC in a dose- and concentration-dependent manner. In order to understand the actual reaction mechanisms involved in the inhibition process, pulse radiolysis investigation of THC with radiolytically produced radicals like hydroxyl, model peroxyl radicals, and azide radicals were done and the transients were detected by kinetic spectrophotometry. The reaction of THC with hydroxyl and azide radicals gave rise to transient absorption in the region 200-400 nm with two peaks at 310 nm and 390 nm. From the spectral properties and kinetics of these radicals, a suitable mechanism is discussed to explain the antioxidant actions of THC.     

Differential antioxidant/pro-oxidant activity of dimethoxycurcumin, a synthetic analogue of curcumin

Dimethoxycurcumin (Dimc), a metabolically stable analogue of curcumin, is under investigation as an anti-tumour agent. Recently a number of studies have been performed on Dimc in this laboratory and also by others. In the present article, all these results have been summarized and wherever possible compared with those of curcumin. Rate constant for reactions of Dimc with superoxide radicals was comparable with that of curcumin, while its reaction with peroxyl radicals was much slower. These results were further supported by the observations on the scavenging of basal ROS levels in lymphocytes and evaluation of antioxidant activities. In line with the earlier reports on curcumin, Dimc was a pro-oxidant and generated ROS in tumour cells. Both curcumin and Dimc were non-toxic to lymphocytes, while exhibiting comparable cytotoxicity to tumour cells. Additionally, these compounds showed higher uptake in tumour cells than in normal lymphocytes. Fluorescence studies on both the compounds revealed their binding to genomic DNA, similar sub-cellular distribution and nuclear localization. All these studies suggested that methylation of the phenolic-OH group in curcumin, although decreasing the antioxidant activity marginally, showed comparable pro-oxidant activity, making it a promising anti-tumour agent.     

Lifetime of peroxyl radicals of poly(U), poly(A) and single-and double-stranded DNA and the rate of their reaction with thiols

Peroxyl radicals of poly(U), poly(A), and single- and double-stranded DNA have been produced by photolysing H2O2 in oxygenated aqueous solution in presence of the substrates. The peroxyl radicals are formed by the reaction of OH radicals with the polynucleotides followed by addition of oxygen. The lifetime of the peroxyl radicals and the rate constant of their reactions with the thiols cysteamine, glutathione and dithiothreithol have been measured by time-resolved e.s.r. spectroscopy. The unusually long lifetimes range from 0.2 to 3.3 s. The activation energy for the decay for all four substrates is 10.3 +/- 1 kcal/mol (43 kJ mol-1). The reaction rate constants with the thiols range from k = 0.8 X 10(4) to 1.3 X 10(5) dm3 mol-1 s-1. The reactions of the thiols with the peroxyl radical of poly(U) are known to prevent strand break formation. This shows that the peroxyl radicals of poly(U) observed by e.s.r. are intermediates in the pathway leading to strand break formation.     

Inactivation of alcohol dehydrogenase (ADH) by ferryl derivatives of human hemoglobin

In this paper, inactivation of alcohol dehydrogenase (ADH) by products of reactions of H2O2 with metHb has been studied. Inactivation of the enzyme was studied in two systems corresponding to two kinetic stages of the reaction. In the first system H2O2 was added to the mixture of metHb and ADH [the (metHb+ADH)+H2O2] system (ADH was present in the system since the moment of addition of H2O2 i. e. since the very beginning of the reaction of metHb with H2O2). In the second system ADH was added to the system 5 min after the initiation of the reaction of H2O2 with metHb [the (metHb+H2O2)5 min+ADH] system. In the first case all the products of reaction of H2O2 with metHb (non-peroxyl and peroxyl radicals and non-radical products, viz. hydroperoxides and *HbFe(IV)=O) could react with the enzyme causing its inactivation. In the second system, enzyme reacted almost exclusively with non-radical products (though a small contribution of reactions with peroxyl radicals cannot be excluded). ADH inactivation was observed in both system. Hydrogen peroxide alone did not inactivate ADH at the concentrations employed evidencing that enzyme inactivation was due exclusively to products of reaction of H2O2 with metHb. The rate and extent of ADH inactivation were much higher in the first than in the second system. The dependence of ADH activity on the time of incubation with ferryl derivatives of Hb can be described by a sum of three exponentials in the first system and two exponentials in the second system. Reactions of appropriate forms of the ferryl derivatives of hemoglobin have been tentatively ascribed to these exponentials. The extent of the enzyme inactivation in the second system was dependent on the proton concentration, being at the highest at pH 7.4 and negligible at pH 6.0. The reaction of H2O2 with metHb resulted in the formation of cross-links of Hb subunits (dimers and trimers). The amount of the dimers formed was much lower in the first system i. e. when the radical forms dominated the reaction of inactivation.     

Differential antioxidant/pro-oxidant activity of dimethoxycurcumin,a synthetic analogue of curcumin

Abstract

Dimethoxycurcumin (Dimc), a metabolically stable analogue of curcumin, is under investigation as an anti-tumour agent. Recently a number of studies have been performed on Dimc in this laboratory and also by others. In the present article, all these results have been summarized and wherever possible compared with those of curcumin. Rate constant for reactions of Dimc with superoxide radicals was comparable with that of curcumin, while its reaction with peroxyl radicals was much slower. These results were further supported by the observations on the scavenging of basal ROS levels in lymphocytes and evaluation of antioxidant activities. In line with the earlier reports on curcumin, Dimc was a pro-oxidant and generated ROS in tumour cells. Both curcumin and Dimc were non-toxic to lymphocytes, while exhibiting comparable cytotoxicity to tumour cells. Additionally, these compounds showed higher uptake in tumour cells than in normal lymphocytes. Fluorescence studies on both the compounds revealed their binding to genomic DNA, similar sub-cellular distribution and nuclear localization. All these studies suggested that methylation of the phenolic-OH group in curcumin, although decreasing the antioxidant activity marginally, showed comparable pro-oxidant activity, making it a promising anti-tumour agent.     

Phenolic chain-breaking antioxidants--their activity and mechanisms of action

This tutorial review is focused on some mechanistic aspects of peroxidation process and chemistry of phenolic chain-breaking antioxidants. Lipids are susceptible to oxidative degradation caused by radicals and during autoxidation (peroxidation) the chain reaction is mediated by peroxyl radicals leading to damage of integrity and the protective and organizational properties of biomembranes. Phenolic antioxidants provide active system of defence against lipid peroxidation, however, the effectiveness of their antioxidant action depends on several important parameters. Stoichiometry of the reaction with free radicals, fate of a phenoxyl radical, polarity of the microenvironment, localization of antioxidant molecules, their concentration and mobility, kinetic solvent effects, and interactions with other co-antioxidants are considered. Principal mechanisms of reaction between phenols and free radicals (Hydrogen Atom Transfer, Proton Coupled Electron Transfer and two mechanisms based on separate electron transfer and proton transfer steps) are described.