Chemical properties of catechols and their molecular modes of toxic action

Environmental Microbiology (2001)3(2),81±91MinireviewChemical properties of catechols and their molecular modes of toxic action in cells,from microorganisms to mammalsNina Schweigert²,Alexander J.B.Zehnder and Rik I.L.Eggen*Swiss Federal Institute for Environmental Science and Technology (EAWAG)and Swiss Federal Institute ofTechnology (ETH),CH-8600Du Èbendorf,Switzerland.SummaryCatechols can undergo a variety of chemical reac-tions.In this review,we particularly focus on complexformations and the redox chemistry of catechols,which play an inportant role in the toxicity of catechols.In the presence of heavy metals,such as iron or copper,stable complexes can be formed.In the presence of oxidizing agents,catechols can be oxidized to semiquinone radicals and in a next step to o-benzoquinones.Heavy metals may catalyse redox reactions in which catechols are involved.Further chemical properties like the acidity constant and the lipophilicity of different catechols are shortly described as well.As a consequence of the chemical properties and the chemical reactions of catechols,many different reactions can occur with biomolecules such as DNA,proteins and membranes,ultimately leading to non-repairable damage.Reactions with nucleic acids such as adduct formation and strand breaks are discussed among others.Interactions with proteins causing protein and enzyme inactivation are described.The membrane±catechol interactions dis-cussed here are lipid peroxidation and uncoupling.The deleterious effect of the interactions between catechols and the different biomolecules is dis-cussed in the context of the observed toxicities,caused by catechols.IntroductionCatechol (1,2-dihydroxybenzene)is used in a variety of applications.It is used as a reagent for photography,dyeing fur,rubber and plastic production and in thepharmaceutical industry (Merck,1989;Milligan and Ha Ègg-blom,1998).Substituted catechols,especially chlorinated and methylated catechols,are by-products in pulp and oil mills (Neilson et al .,1991;Capasso et al .,1995).Catechols are intermediary products from the degradation of aromatic compounds and lignin by microorganism (Crawford,1981;van der Meer et al .,1992).In humans and mammals,catechols can occur as metabolites in the degradation of benzene or estrogens (Porteous and Williams,1948;Bolton et al .,1998)or as endogenous compounds,such as neurotransmitter and their precursors [adrenaline,noradrenaline,dopamine and L-DOPA (L-3,4-dihydroxy-phenylanaline)](AndeÂn,1979).Additionally,catechols can be taken up in the form of tabacco smoke (as catechol,catechol semiquinones and polymerized catechols)(Stone et al .,1995;Pryor et al .,1998)or as food components (e.g.catechol,dopamine,caffeic acid,tea catechin)(Oshima,1965;Tressl et al .,1978;Hayakawa et al .,1997;Moran et al .,1997).In 1950,it was suggested that benzene metabolites were responsible for the benzene toxicity in mammals and humans (Dustin,1950)and according to Porteous and Williams (1948)catechol was already identified as one of these metabolites at the end of last century.The toxicity of catechols for microorganisms has been demonstrated in the past years (Fritz et al .,1992;HellmeÂr and Bolcsfoldi,1992;Capasso et al .,1995;Boyd et al .,1997),and has been suggested to be the reason for the difficulties in cultivating microorganisms on benzene,toluene or chlorobenzene (Fritz et al .,1992).Several studies additionally indicated the toxicity of catechols for water flea,zebra fish,trout,rabbit,cat,rat,mouse and for human cell lines (Garton and Williams,1948;Hattula et al .,1981;Pellack-Walker et al .,1985;Neilson et al .,1991;Svenson and Hynning,1997;Rahouti et al .,1999;Van Den Heuvel et al .,1999).Indeed,in the environment toxic concentrations of catechols have been found (Neilson et al .,1991;Capasso et al .,1995).Despite the fact that catecholsQ 2001Blackwell Science Ltd²Present address:Solvias AG,Postfach 4002,Basel,Switzerland.*For correspondence.E-mail eggen@eawag.ch;Tel.(141)18235320;Fax (141)18235547.Received 28August,2000;revised 19December,2000;accepted 3January,2000.are ubiquitous,and their toxicity has been observed in a variety of organisms the modes of action causing the toxicity are hardly understood.In the following review,an overview about the chemical properties of catechols is given,which is required to understand their possible toxic modes of action.The molecular modes of action found for catecholic compounds are summarized and discussed,subsequently.Because basic toxic processes in the context of catechol toxicity are similar in different cell types,ranging from microorganisms to mammals,the information from reactions in different cell types is integrated.Many concepts have,however,been developed and validated with the help of microorganisms as model systems.The chemical reactions and molecular modes of action,treated in this review,are depicted in Fig.1.Finally,an attempt is made to link the observed toxicities to the different modes of action.Chemical properties of catecholCatechols can form stable complexes with various di-and trivalent metal ions,the complexes with trivalent ions being the most stable (Table 1).Catechols can alsoundergo redox reactions,cycling between catechols,semiquinone radicals and ortho-benzoquinone (Fig.2).Complex formationThe complexation of catechol has been studied most intensively with iron (Avdeef et al .,1978;Hider et al .,1981).Depending on the pH,a number of mono-,di-,or tri-coordinated complexes are formed:Mono ÿcoordinated complexes: Fe II cat ´2 H 2O 4 1 Fe III cat 2± H 2O 4 1Di ÿcoordinated complexes: Fe II cat X ± cat 2 OH 2 2 Fe III cat 2± cat 2 OH 2 2Tri ÿcoordinated complexes:Fe II cat´ cat 2 2 0 Fe III cat 2± 3 3±The different abbreviations for the different forms of catechols used in the text are:cat for catechol;cat 2for the mono-deprotonated catechol;cat 2±for the di-depro-tonated catechol,cat´for the semiquinone radical and cat 2for the mono-deprotonated semiquinoneradical.Fig.1.Chemical reactions of catechols in the environment and in cells and molecular modes of action in cells,treated in this review.Chemical reactions:1,Complex formation with heavy metals;2,Redox cycling;3,Production of reactive oxygen species (ROS)in the reaction with heavy metals and oxygen.Molecular modes of actions in cells:4,DNA damage (for example,strand breaks and DNA adduct formation);5,protein damage (for example,protein cross-linking via disulphide groups);6,absorption in membranes and possible interactions.82N.Schweigert,A.J.B.Zehnder and R.I.L.EggenQ 2001Blackwell Science Ltd,Environmental Microbiology ,3,81±91Fe(II)complexes form preferentially at acidic pH,whereas Fe(III)occurs at pH .7.The type of iron±catechol complex present depends on the iron/catechol ratios.Mono-coordinated complexes form preferentially at higher ratios,whereas tri-coordinated complexes are dominant at lower ratios (Hider et al .,1981).The existence of an (iron(III)2-(catchol)4complex has also been suggested (Hider et al .,1981).Due to the high stability of catechol±metal complexes,compounds con-taining catecholic nuclei can sequester metals from other complexes,thus preventing metals from undergoing redox reactions (Avdeef et al .,1978).Redox chemistryThe equilibrium potential for the one-electron transfer between catechol and the semiquinon radical (cat´)is 100mV at pH 7(Eberson,1985).For the second electron transfer between the semiquinon radical and the catechol dianion (cat 2±),an equilibrium potential was reported of 530mV,139mV and 43mV for pH 7,pH 11and pH 13.5respectively (Steenken and Neta,1979).As a consequence,under physiological conditions catechol is not auto-oxidized (Irons and Sawahata,1985),except in the presence of heavy metals (e.g.copper)(Irons and Sawahata,1985;Schweigert et al .,2000).Catechol complexes with iron also prevent the metal from undergoing redox reactions as in the case of di-coordinated iron (III)complexes.In contrast,mono-coordi-nated complexes allow iron to be involved in electron transfer reactions (Avdeef et al .,1978).Several enzymes in plants,animals and bacteria are able to catalyse the oxidation of catechol to benzoqui-none,such as the copper-containing fungal laccase andsuperoxide dismutase,some haem-containing peroxi-dases and tyrosinase (Mason et al .,1961).When catechol is oxidized enzymatically or in the presence of oxygen and heavy metals,one electron is transferred to molecular oxygen,consequently superoxide (O 22)is formed.In the presence of heavy metals (e.g.copper,iron),superoxide is further reduced to hydrogen peroxide (H 2O 2)and hydroxyl radicals (´OH).These reactive oxygen species (ROS)can be harmful to cells and organisms,if they are not eliminated (Mason et al .,1961;Schweigert et al .,2000).Further chemical propertiesThe acidity dissociation constants (p K a )for catechol are both above pH 7:p K a (1),9.25(Schweigert et al .2001);p K a (2),13.0(Dean,1985).The acidity dissociation constant for the catechol radical can,however,be much lower.Patel and Wilson (1973)determined that the p K a (1)of the hydroquinone radical is pH 4.1,and pH 9.85for hydroquinone.Thus,catechol occurs in the non-disso-ciated form and the catechol radical most probably in the dissociated form at physiological pH.The partitioning coefficients of catechol in an n-octanol water system (K ow )and in a membrane-water system (K mw )are 7.85and 4.47respectively (Schweigert et al .,2001).Catechol is not very lipophilic and does not accumulate considerably in membranes or fatty tissues.Alkylated or halogenated catechols,however,have higher partitioning coefficients and will therefore accumulate in lipids.The K ow for tetrachlorocatechol is,for example,11400(Schweigert et al .,2001).Molecular modes of action of catechols in cells Catechols can act both as antioxidant,preventing lipid peroxidation,and as pro-oxidant damaging macromole-cules such as DNA and proteins.Catechols can also destroy membrane functioning due to their redox cycling activity.In this section,some possible modes of action are summarized.Genotoxicity by ROS production and DNA adduct formationCatechol as such does not cause oxidative DNA damage in vitro .Combined with heavy metals (e.g.Cu 21,Fe 31)Table 1.Stability of metal±catechol complexes at 258C.Metal ion Equilibrium Log K Ionic strength Fe(II)ML/M 1L 7.95 1.0ML 2/M 1L 213.5 1.0Fe(III)ML/M 1L 20.00.1ML 2/M 1L 234.70.1ML 3/M 1L 343.80.1Cu(II)ML/M 1L 13.90.1ML 2/M 1L 224.90.1Al(III)ML/M 1L 16.30.1ML 2/M 1L 229.30.1ML 3/M 1L 3370.1M,is the metal ion;L,corresponds to the ligand catechol,data derived from Martell and Smith (1982,1989).Fig.2.Redox cycling of catechol.Thereactions can,for example,be catalysed by heavy metals.Molecular modes of catechol toxicity83Q 2001Blackwell Science Ltd,Environmental Microbiology ,3,81±91and in the presence of molecular oxygen,DNA strand breaks can be observed(Li and Trush,1994;Schweigert et al.,1999).DNA strand breaks are caused by a redox reaction of Cu(II)and catechol to yield Cu(I)and the semiquinone radical and a subsequent copper catalysed reduction of molecular oxygen,where superoxide and hydrogen peroxide are formed.A DNA±copper±oxo complex[DNA-Cu(I)-OOH]finally causes the DNA strand breaks by splitting of hydroxyl radicals in the vicinity of the DNA(Fig.3)(Schweigert et al.,2000).Different plant phenolics with catechol nuclei,such as caffeic acid,protocatechuic acid and chlorogenic acid stimulate the DNA degradation induced by Fenton reagents,e.g.Fe(III)and bleomycin(Hiramoto et al.,1996;Moran et al.,1997).LeÂvay et al.have shown that the neurotransmitter dopamine which has a catechol nucleus and is found as an endogenous compound in plants and mammals causes DNA damage in two different ways:DNA adducts are formed and oxidative DNA damage upon incubation with copper occurs(LeÂvay et al.,1997).The DNA damage caused by the catechol and hydroquinone containing fraction of aqueous cigarette tar(ACT)has also been investigated for its DNA damaging activity in in vitro experiments.Hydrogen peroxide,superoxide and hydroxyl radicals are produced by this fraction and cause oxidative DNA damage(Pryor et al.,1998).This ACT also contains the tarradical,Fig.4.(A)DNA adduct formation by the reaction of catechol estrogens with guanidines(Akanni and Abul-Hajj,1997).(B)Formation of8-hydroxy-2H-deoxyguanisine(8-oxo-dG)by the reaction of guanisine with hydroxyl radicals(Halliwell and Dizdaroglu,1992).Fig.3.Production of DNA degrading hydroxyl radicals in the presence of oxygen,copper and DNA.84N.Schweigert,A.J.B.Zehnder and R.I.L.EggenQ2001Blackwell Science Ltd,Environmental Microbiology,3,81±91consisting of polymerized catechol,suggesting that catechol is involved in the production of the ROS.The tar radicals bind to DNA and DNA adducts are formed (Stone et al .,1995).The DNA damaging activity of catechol estrogens (CE)has been intensively studied.During the redox cycling of CEs,in the presence of copper(II),semiquinones and reactive oxygen species are formed and an increased rate of oxidative DNA damage can be measured (Seacat et al .,1997;Mobley et al .,1999).Semiquinones and/or qui-nones formed from CEs can also damage DNA directly by covalent binding to the DNA (Dwivedy et al .,1992;Akanni et al .,1997).This catechol moiety is responsible for the formation of CE-DNA adducts (Akanni et al .,1997;Bolton et al .,1998)as shown for guanidine in Fig.4A.In vivo,DNA damage experiments have been per-formed with bacteria and mammals.A peroxide evolution,indicating a redox cycling of catechol,was measured in Escherichia coli cells,exposed to catechol or a combina-tion of catechol and copper (Schweigert et al .,1999).Despite of the peroxide production,catechol is not mutagenic in E.coli and Salmonella typhimurium,when tested alone or in combination with copper (Haworth et al .,1983;Schweigert et al .,1999).The absence of muta-genicity is probably due to activated defence systems as for example an induction of a catalase.Catalases reduce hydrogen peroxide to water and molecular oxygen and also detoxify organic peroxides (Schweigert et al .,1999).Therefore,no DNA±copper±oxo complexes are formed and no additional DNA damage occurs.Catechol alone does not cause oxidative DNA damage in bone marrow as shown by Kolachana et al .(1993).In combination withphenol or hydroquinone (neither compound induces oxidative DNA damage alone),however,the 8-hydroxy-2H -deoxyguanisine (8-oxo-dG)level increases,which is indicative for the occurrence of oxidative DNA damage (Kolachana et al .,1993).In Fig.4B,the formation of 8-oxo-dG is shown in a reaction of DNA with hydroxyl radicals.LeÂvay and Bodell (1996)measured DNA adduct formation in the human cell line HL-60.In this cell line,catechol forms three different non-identified DNA adducts.A treatment of HL-60cells with catechol raises the internal hydrogen peroxide concentration as also seen in E.coli cells.The production of the DNA adducts depends on a peroxidase activity,it also increases when the hydrogenperoxide concentration is added to the cultures (LeÂvay et al .,1993).Thus,the cellular hydrogen peroxide level seems to regulate the peroxidase-dependent activation of catechol to form DNA adducts.The authors suggest the following mechanism:the peroxidase reduces hydrogen peroxide with catechol as a reducing agent.As a consequence,reactive catechol intermediates are formed leading to the DNA adduct formation.It is not specified whether the semiquinone radical or the o -quinone are the reactive intermediate forming the DNA adducts.Catechol has also been shown to have further DNA damaging activities,for example,it can cause chromatid strand breaks,chromosome aberrations in metaphase and chromatid exchanges (Morimoto and Wolff,1980;Hecht et al .,1981;Stich et al .,1981;Erexson et al .,1985).Again,an activation of catechol has in two cases been proven to be necessary (Hecht et al .,1981;Stich et al .,1981).Furthermore,catechol can act as co-carcinogen,whenFig.5.Three possible forms of catechol±protein interactions.A.Reaction of oxidized catechol with sulphydryl groups (Bolt and Kappus,1974).B.Reaction of oxidizedcatechol with amino groups,for example the 1-amino group of lysine (Gottlieb and Wurzel,1974).C.Reduction of the semiquinone radical by a sulphydryl group and subsequent protein cross-linking in the presence of oxygen (Stoyanovsky et al .,1995).Molecular modes of catechol toxicity 85Q 2001Blackwell Science Ltd,Environmental Microbiology ,3,81±91combined with various carcinogens (Hecht et al .,1981;Atchison et al .,1982;Mirvish et al .,1985).Catechol exhibits inhibitory effects on the DNA synth-esis in the mouse lymphoma cell line L5178YS.These effects have been explained by DNA damage caused by DNA alkylation or oxidative DNA damage (Pellack-Walker et al .,1985).But similar effects in human T lymphoblasts have been explained by enzyme inhibition (see below).In an animal model of hormone-mediated carcinogen-esis,male golden Syrian hamsters developed renal carcinoma upon prolonged exposure to 17b -estradiol (Butterworth et al .,1997).This phenomenon has been linked with hormonal potency and/or metabolites of estro-gens.The major metabolites formed by aromatic hydro-xylation of the estrogen are catechol estrogens,of which the catechol forms the reactive moiety (oxidation to ortho -quinone and subsequent redoxcycling with the generation of reactive oxygen species).In addition,can quinones react with cellular nucleophiles such as glutathione (GSH),and quinone-thioethers,with a variety of toxic activities being formed (Monks and Lau,1992).In the Syrian hamsters,catechol estrogen GSH conjugates have been formed at doses equivalent to those causing estrogen-induced carcinogenicity (Butterworth et al .,1997).No direct link has,however,be established yet between the catechol estrogen GSH conjugates and nephrocarcinogenicity.In summary,all catecholic compounds investigated can cause DNA damage in vitro or in vivo ,upon activation,for example,by heavy metals,or by cellular metabolism and conjugation reactions.DNA damage is either due to DNA-adduct formation by the catechols or their reaction intermediates or it is due to the formation of ROS causingan oxidation of DNA bases and/or DNA strand breaks.However,genotoxicity is not the reason for the toxicity of catechols in E.coli (Schweigert et al .,1999).But we can not exclude that the potency of catechols to cause a variety of DNA damages leads to chronic effects by a slow accumulation of mutations,which are not visible in bacterial mutagenicity assays.Protein inactivationOxidized catechols react with sulphydryl groups of proteins and glutathione.Catechol thereby binds either directly to sulphydryl groups and inactivates the protein,or protein and glutathione radicals are produced.These radicals might subsequently lead to protein cross-linking and glutathione dimer formation (Stoyanovsky et al .,1995).As glutathione is an important reductant in cells,a dimer formation can change the redox status of the cell,thereby producing oxidative stress (Halliwell and Gutteridge,1989).Catechol inhibits the rate limiting step of DNA synthesis in human T lymphoblasts,probably by inhibiting the ribonucleotide reductase.A covalent binding of catechol to the protein does not cause this inhibition or a protein cross-linking as the addition of FeCl 3reverses the effect.The formation of iron±catechol complexes seems to be the reason for the observed inhibition (Li et al .,1997).In other cases,where catechol inhibits enzyme func-tioning,the mode of action is not understood yet.In brush border membrane vesicles,isolated from rat intestine,catechol inhibits the intestinal sucrase (Welsch et al .,1989).The results were independent of the oxidation state of catechol.Cytochrome P4502E1,responsible fortheFig.6.Model for the active electron transport in membranes caused by higher chlorinated catechols.The different catechol species (H 2cat m ,Hcat 2m and [catH 2catH]2m ,the neutral catechol molecule,the catechol anion and a heterodimer,consiting of a catechol molecule and a catechol anion)are absorbed in the polar regions of the membrane and H 2cat and Hcat 2are in equilibrium between the aqueous phase (w)and the membrane (m).After building up an electrochemical gradient,the charged species Hcat 2m and [catH 2catH]2m migrate (mig.)in the charge gradient.The diffusion (diff.)of H 2cat from the positively to the negatively charged side re-establishes the catechol equilibrium between the polar regions of the membrane.86N.Schweigert,A.J.B.Zehnder and R.I.L.EggenQ 2001Blackwell Science Ltd,Environmental Microbiology ,3,81±91oxidation of benzene in rat and human liver microsomes,is inactivated by the oxidation products of catechol,either the semiquinone radical or o -quinone (Gut et al .,1996).For the inactivation of the sucrase and cytochrome P4502E1,a protein cross-linking is the most probable explanation for the enzyme inactivation.Compounds with catechol moieties,such as CEs,L-DOPA,dopamine and a -methyl-DOPA can also cross-link proteins (Bolt and Kappus,1974;Gottlieb and Wurzel,1974;Scheulen et al .,1975;Freyberger and Degen,1989)for example,L-DOPA and dopamine cross-link neurofilaments.The reaction is accelerated by the addition of the (redox cycling)heavy metals copper,iron and manganese,suggest-ing that an oxidized form of the catecholic compounds isresponsible for cross-linking.It has been shown that not only the sulphydryl groups,but also the 1-aminogroups of lysine react with the oxidized catechols (Montine et al .,1995).Protein damage by cross-linking,such as reactions with sulphydryl groups or 1-amino groups,is observed upon exposure to all catecholic compounds,investigated in this respect.In Fig.5different interactions of catechols with proteins are summarized.Uncoupling leading to a destruction of the membrane potential;effects of complex formationsThe deleterious effect of catechols on membranes is not caused by the production of ROS or reactions withlipidFig.7.A.A possible chain reaction during lipid peroxidation in fatty acids,induced by hydroxyl radicals.B.A possible chain breaking reaction,due to the transfer of a proton and electron from catechol to the fatty acid radical.Molecular modes of catechol toxicity 87Q 2001Blackwell Science Ltd,Environmental Microbiology ,3,81±91molecules,it is due to the destruction of the membrane potential in energy transducing membranes.Catechol shows inhibitory effects on the oxidative phosphorylation in rat liver mitochondria,with an I50(concentration causing 50%inhibition)of2mM and it is thereby more active than phenol(I50 5mM)(Mitsuda et al.,1963).Depending on their concentration,catechols stimulate the resting respiration(at lower concentrations),show no measurable effect(intermediate concentrations)or inhibit the respira-tion(at high concentrations),these effects are identical to those observed for chlorinated phenols,but they are less pronounced in the case of catechol(Mitsuda et al.,1963). In membrane vesicles,isolated from the photosynthetic bacterium Rhodobacter sphaeroides,an uncoupling of the electron transport from the ATP-synthesis has been shown using photosynthetically active membranes (Schweigert et al.,2001).Millimolar concentrations of catechol and4-chlorocatechol are needed for a significant effect.These catechols presumably act as narcotics, passively destroying the membrane potential by a perturbation of the lipid bilayer.Higher chlorinated catechols,by contrast,actively destroy the membrane potential by transporting electrons over the membrane (Fig.6).Already micromolar concentrations of these catechols showed clear effects.The concentrations of the individual catechols causing membrane toxic effects are in agreement with the concentrations causing toxicity in binations of the different catechols with copper were investigated with this method.In the case of the higher chlorinated catechols,copper strongly inhibits the uncoupling,while it augments the membrane toxicity of the lower chlorinated catechols.This is again in agreement with the toxicity determined in E.coli(Schwei-gert et al.,2001).The conformity strongly suggests that the uncoupling of the electron transport due to narcosis or active electron transport is the mode of action responsible for the observed toxicity.Inhibition of lipid peroxidationBeside all the negative effects catechol can have in cells, catechols also have advantageous effects:they inhibit lipid peroxidation(LPO).Lipid peroxidation,the oxidative modification of poly-unsaturated fatty acids,is a chain reaction,which can be initiated by a variety of oxidizing compounds(Fig.7a).It is a major contributor to membrane damage and seems to be the cause of a range of pathological processes(Dix and Aikens,1993).Catechol and catechol derivatives, such as CEs,adrenaline,adrenergic catechol derivatives (e.g.dopamine,isoproterenol),catechins and caffeic acid have been shown to strongly inhibit LPO.The lipid fraction of the test systems used included liposomes,micro-somes,hepatocytes or mitochondria,and also LPO-initiating agents tert-butyl hydroperoxide,ferrous iron, CCl4,NADPH,ferrous iron1ascorbate,ferrous iro-n1ADP,ferric iron1ADP1adriamycin have been used(Kappus et al.,1977;Nakano et al.,1987;Masaki et al.,1989;Uchida et al.,1996;Hayakawa et al.,1997; Miura et al.,1998).Most of these compounds or mixtures cause LPO indirectly,by a production of ROS,which oxidize the unsaturated l4,however,might react with the lipids itself,after an activation to the radical´CCl3 (Kappus et al.,1977).Different explanations for the antioxidant activity of catecholic compounds in LPO assays can be found in the literature.Chelation of iron is one possible explanation for the inhibition of LPO induced by iron-containing systems(Gilbert and Sawas, 1983;Braughler et al.,1988;Moran et al.,1997).It has also been discussed if the inhibition of LPO induced by ferrous iron1ADP or CCl4in liver microsomes can be explained by an inhibitory effect of catechol on cyto-chrome c reductase.This enzyme catalyses LPO, induced by the combination of ferrous iron1ADP,and is assumed to be responsible for the activation of CCl4to the´CCl3radical(Kappus et al.,1977).Nakano et al. (1987)has found that CEs inhibit LPO in a microsomal system,induced by ferric iron,ADP and adriamycin.They suggest that CE±iron complexes compete with iron±ADP±adriamycin complexes for cytochrome P450,a key enzyme for iron-induced LPO.This competition as well as iron chelation and inhibitory effects on enzymes might be reasons for a reduction of LPO by catechol.However,the fact that catechol and its derivates inhibit LPO in all systems tested suggests that the mechanism is probably the same in all(Kappus et al.,1977;Uchida et al.,1996). The most reasonable explanation is,therefore,that the free radical scavenging properties of catechol and its derivates,due to their ability to form stable radicals,are responsible for the inhibitory effect as shown in Fig.7b. Concluding remarksCatecholic compounds belong to a heterogenous group of reactive molecules,but their reactivity is often determined by the catechol nucleus.In this overview,we have attempted to convey an appreciation of the complexity of the chemistry and biological behaviour of catecholic compounds.Because catechols readily undergo oxidation to form semiquinone radicals and quinones,which are in some aspects more reactive than the catechols,the deleterious effects of these species has also been considered.The mechanisms most frequently cited to explain the toxicity of catecholic compounds are:(i)the generation of ROS by redox reactions;(ii)DNA damage in the form of oxidative damage or DNA arylation;(iii)protein damage by sulphydryl arylation or oxidation;and(iv) interference with electron transport in energy transducing88N.Schweigert,A.J.B.Zehnder and R.I.L.EggenQ2001Blackwell Science Ltd,Environmental Microbiology,3,81±91。