Biological Reactive Intermediates Research Articles

Dopamine-derived biological reactive intermediates and protein modifications: Implications for Parkinson's disease

Dopamine (DA) undergoes monoamine oxidase catalyzed oxidative deamination to 3,4-dihydroxyphenylacetaldehyde (DOPAL), which is metabolized primarily to 3,4-dihydroxyphenylacetic acid (DOPAC) via aldehyde dehydrogenase (ALDH). Previous studies demonstrated DOPAL to be neurotoxic, more so than DA and other metabolites, and implicated the aldehyde intermediate as a factor in the pathogenesis of Parkinson's disease (PD). However, the mechanism for generation of DOPAL at aberrant levels and the pathways for toxicity are not conclusively known. Various models for DA catabolism revealed the susceptibility of DOPAL biotransformation (e.g., ALDH) to products of oxidative stress, e.g., 4-hydroxy-2-nonenal, at physiologic/pathologic levels and agents that induce oxidative stress. An elevated concentration of DOPAL correlated with increased protein modification with subsequent work demonstrating significant reactivity of the DA-derived electrophile toward protein nucleophiles compared to DA and other metabolites, e.g., DOPAC. The addition of DOPAL to proteins proceeds via reaction of the aldehyde with Lys residues, yielding a Schiff base; however, post-adduction chemistry occurs for the DOPAL-modification resulting in protein cross-linking. Preliminary work indicates enzymes in DA synthesis and catabolism to be cellular targets for DOPAL. Functional consequences for elevated levels of the DA-derived aldehyde and protein modification may include adverse cellular effects. These data implicate DOPAL as a toxic and reactive intermediate potentially serving as a “chemical trigger” for some stage of PD pathogenesis.

Flavonoids and alkenylbenzenes: New concepts in bioactivation studies

The present paper focuses on the biological reactive intermediates formed from two categories of botanical ingredients: flavonoids and alkenylbenzenes. The paper especially presents an overview of three concepts in bioactivation studies on flavonoids and alkenylbenzenes elucidated by our recent studies. These new concepts include (i) the fact that reactive electrophilic quinone/quinone methide type metabolites of flavonoids may be the intermediates required for the induction of the beneficial gene expression through electrophile responsive element (EpRE)-mediated pathways, pointing at a possible beneficial effect of a reactive intermediate, (ii) the development of physiologically based kinetic (PBK) and physiologically based dynamic (PBD) models providing a new way to obtain insight in levels of formation of biologically reactive and unstable intermediates in vivo at high but also more realistic low dose levels, and (iii) the concept of the matrix effect that should be taken into account when studying the bioactivation of food-borne genotoxic carcinogens including the alkenylbenzenes, the bioactivation of which was shown to be inhibited by flavonoids. Together the results presented reveal that by studying the mode of action (MOA) new concepts in bioactivation studies of importance for future risk assessment and/or risk-benefit assessment of the flavonoids and alkenylbenzenes are obtained.

Involvement of mitochondrial/lysosomal toxic cross-talk in ecstasy induced liver toxicity under hyperthermic condition

The initial objectives of this study were to evaluate the extent of 3, 4-methylenedioxymethamphetamine (MDMA) induced loss of cell viability (cytotoxicity), induction of reactive oxygen species formation and damage to sub-cellular organelles (e.g. mitochondria/lysosomes) in freshly isolated rat hepatocytes under normothermic conditions (37 °C) and to compare the results with the effects obtained under hyperthermic conditions (41 °C). MDMA induced cytotoxicity, reactive oxygen species formation, mitochondrial membrane potential decline and lysosomal membrane leakiness in isolated rat hepatocytes at 37 °C. A rise in incubation temperature from 37 °C to 41 °C had an additive/synergic effect on the oxidative stress markers. We observed variations in mitochondrial membrane potential and lysosomal membrane stability that are significantly (P < 0.05) higher than those under normothermic conditions. Antioxidants, reactive oxygen species scavengers, lysosomal inactivators, mitochondrial permeability transition (MPT) pore sealing agents, NADPH P450 reductase inhibitor, and inhibitors of reduced CYP2E1 and CYP2D6 prevented all MDMA induced hepatocyte oxidative stress cytotoxicity markers. It is therefore suggested that metabolic reductive activation of MDMA by reduced cytochrome P450s and glutathione could lead to generation of some biological reactive intermediates which could activate reactive oxygen species generation and cause mitochondrial and lysosomal oxidative stress membrane damages. We finally concluded that hyperthermia could potentiate MDMA induced liver toxicity probably through a mitochondrial/lysosomal toxic cross-talk in freshly isolated rat hepatocytes.

Biological reactive intermediates that mediate dacarbazine cytotoxicity

In the following, the cellular and molecular mechanism of cytotoxicity induced by prodrug dacarbazine toward the isolated rat hepatocytes was studied. Accelerated cytotoxicity screening technique (ACMS) was used to perform this study. Addition of dacarbazine to isolated rat hepatocytes resulted in reactive oxygen species (ROS) formation, and lysosomal membrane leakiness before hepatocyte lysis occurred. Hepatocyte ROS generation was inhibited by desferoxamine (a ferric chelator). Cytotoxicity was prevented by antioxidants or ROS scavengers (mannitol or dimethylsulfoxide), cytochorome P450 inhibitors (phenylimidazole, diphenyliodonium chloride, 4-methylpyrazole, and benzylimidazole). In addition to lysosomal damage, dacarbazine caused hepatocyte protease activation and cell proteolysis. Dacarbazine cytotoxicity is associated with ROS (H(2)O(2), O(2)(*-) ) generation. It is suggested that H(2)O(2) could cross the lysosomal membrane, react with lysosomal Fe(2+) to form hydroxyl radical (Haber-Weiss reaction) which is the major cause of lysosomal membrane leakiness, proteases, and other digestive enzymes' release and finally the cell death.

Cytochrome P450 Oxidation of the Thiophene-Containing Anticancer Drug 3-[(Quinolin-4-ylmethyl)-amino]-thiophene-2-carboxylic Acid (4-Trifluoromethoxy-phenyl)-amide to an Electrophilic Intermediate

Compounds that are enzymatically transformed to reactive intermediates are common in nature. Some drugs and many phytochemicals that contain a thiophene ring are oxidized by cytochrome P450 to biological reactive intermediates (BRI) that can covalently bind to thiol nucleophiles. The investigational anticancer agent 3-[(quinolin-4-ylmethyl)-amino]-thiophene-2-carboxylic acid (4-trifluoromethoxy-phenyl)-amide (OSI-930) contains a thiophene moiety that can be oxidized by P450s to an apparent sulfoxide, which can react via Michael-addition to the 5-position of the thiophene ring, as demonstrated by mass spectral characterization of several thioether conjugates of the presumed thiophene S-oxide. Furthermore, a stable deuterium isotope retention experiment in which solvent deuterium was incorporated into the thiophene verifies the sulfoxide pathway. Various thiol nucleophiles are shown by tandem mass spectra to bind with this BRI, which is activated by P450 3A4 and to a slight degree, P450 2D6. Yet various safe drugs, phytochemicals, and endogenous molecules, all noted for their activation to BRI, are not toxic at a normal dose. Thus, multiple features determine any consequence of a BRI, with these complexities determining why one BRI is benign while another is not. The retention of covalent protein adducts of radio-labeled intermediate rat tissue has a half-life of about 1-1.5 days; hence, modified protein is cleared and replaced relatively quickly.

Many Drugs and Phytochemicals Can Be Activated to Biological Reactive Intermediates

The metabolism of drugs and other xenobiotics is exemplified by cytochrome P450-mediated oxidation to more hydrophilic compounds. Enzymatic oxidation of some functional groups, however, can result in transient reactive intermediates--a transformation that is common in nature. Some drugs and many phytochemicals that contain, for example, a thiophene ring are oxidized by cytochrome P450 to reactive intermediates, such as sulfoxides, that can covalently bond to thiol nucleophiles in macromolecules, such as proteins. Many other functional groups can be oxidized to reactive intermediates--for instance, olefins, alkynes, alkylamines, furans, and p-aminophenols. Because any consequence of a biological reactive intermediate (BRI) is dependent on many factors a number of BRIs are benign. Toxicity is determined by complex and specific arrays of parallel and/or serial pathways and cellular states, not by entities or individual reactions. Because the formation of reactive intermediates can correlate with unacceptable toxicity, it is important to understand if or how a compound may undergo this type of transformation. Not all substances that form reactive intermediates are toxic; many are not. Therefore, it is critical to understand the mechanism of metabolism in considering any toxicity relevance and in evaluating animal models. Indeed, the complex nature of these many interactions and entities underscores the fact that the presence of a BRI is not an accurate indicator of human toxicity. A BRI is neither good nor a priori bad. It is a potential detoxication product and an incidental fate of the xenobiotic.

Error-prone Translesion Synthesis by Human DNA Polymerase η on DNA-containing Deoxyadenosine Adducts of 7,8-Dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene

When human DNA polymerase eta (pol eta) encounters N6-deoxyadenosine adducts formed by trans epoxide ring opening of the 7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BaP DE) isomer with (+)-7R,8S,9S,10R configuration ((+)-BaP DE-2), misincorporation of A or G and incorporation of the correct T are equally likely to occur. On the other hand, the enzyme exhibits a 3-fold preference for correct T incorporation opposite adducts formed by trans ring opening of the (-)-(7S,8R,9R,10S)-DE-2 enantiomer. Adducts at dA formed by cis ring opening of these two BaP DE-2 isomers exhibit a 2-3-fold preference for A over T incorporation, with G intermediate between the two. Extension one nucleotide beyond these adducts is generally weaker than incorporation across from them, but among mismatches the (adducted A*) x A mispair is the most favored for extension. Because mutations can only occur if mispairs are extended, this observation is consistent with the occurrence of A x T to T x A transversions as common mutations in animal cells treated with BaP DE-2 isomers. Adducts with S absolute configuration at the point of attachment of the hydrocarbon to the base inhibit incorporation and extension by pol eta to a lesser extent than their R counterparts. Template-primers containing each of the four isomeric dA adducts derived from BaP DE-2 and two adducts derived from 9,10-epoxy-7,8,9,10-tetrahydrobenzo-[a]pyrene in which the 7- and 8-hydroxyl groups of the DEs are replaced with hydrogens exhibit reduced electrophoretic mobilities relative to the unadducted oligonucleotides. This effect is largely independent of DNA sequence. Decreased mobility correlates with an increased rate of incorporation by pol eta, suggesting a systematic relationship between the overall DNA structure and efficiency of the enzyme.

Biological Reactive Intermediates VII: BRIs and Human Health and Disease Westin La Paloma Resort, Tucson, Arizona January 4−7, 2006

Biological Reactive Intermediates VII: BRIs and Human Health and Disease Westin La Paloma Resort, Tucson, Arizona January 4−7, 2006

Mechanistic and toxicokinetic data reducing uncertainty in risk assessment

Risk characterization comprises hazard identification describing the intrinsic toxic potential of a chemical, knowledge of dose-response-relationships, as well as of toxicokinetics describing quantitatively the relation between external and internal dose and exposure assessment. Compounds that induce reversible effects, which are repaired during and after exposure, are considered thresholded and allow definition of a NOEL. Biological reactive intermediates of chemicals have the potential to bind covalently to cellular macromolecules like proteins and DNA. Such interaction may not be repaired completely. If damage is not repaired, the effect persists and accumulates upon repeated exposure. In such cases a NOEL cannot be determined. Thus, in the risk assessment process, data on covalent binding (CB) are of qualitative and together with toxicokinetics of quantitative significance. Qualitatively, CB, especially with DNA and in correlation with this to proteins, is indicative for an irreversible and non-thresholded mutagenic and carcinogenic effect. Absence or presence of CB assists to differentiate between primarily genotoxic and thresholded non-genotoxic carcinogens. Quantitatively, toxicokinetics together with CB are used to quantify internal exposure and target dose, which is a prerequisite for species–species extrapolation, and for extrapolation from high dose to low dose. For example, the toxicokinetics of the reactive intermediates of styrene and ethylene have been determined in rodents and humans and modeled to predict dose-responses of internal exposure. It is described in this communication that such information, together with other parameters like cell proliferation as a result of cytotoxicity, is the basis for quantitative risk assessment of human exposure to these compounds. Also for chlorobenzene, the relevance of toxicokinetics for estimating the human health risk is demonstrated.

Mechanisms of Carcinogenicity of Aryl Hydrazines, Aryl Hydrazides, and Arenediazonium Ions

Aryl hydrazines carcinogenesis has been studied for over 25 years and remains poorly understood, although most aryl hydrazines are toxic, tumorigenic, or carcinogenic. In this article, aryl hydrazine carcinogenesis is reviewed comprehensively. The relevant chemistry and biochemistry of aryl hydrazines are first addressed and provide the framework for understanding how aryl hydrazines are metabolized, the reactive intermediates that are produced, and the biological reactive intermediates and products that are formed. Issues of DNA damage, mutagenicity, and enzyme activation are next addressed followed by a brief review of aryl hydrazine tumorigenicity studies. Because several related substrates are metabolized to the same intermediates as are aryl hydrazines, they are briefly discussed. The review concludes with a short discussion of the possible mechanism of carcinogenesis by aryl hydrazines.

Biological reactive intermediates and mechanisms of cell death.

Apoptosis and necrosis are two modes of cell death with distinct morphological and biochemical features. Apoptosis is an active process characterized by cell shrinkage, nuclear and cytoplasmic condensation, chromatin fragmentation and phagocytosis. In contrast, necrosis is a passive form of cell death associated with inflammation resulting from cellular and organelle swelling, rupture of the plasma membrane and spilling of cellular contents into the intercellular milieu. Lethal levels of biological reactive intermediates may trigger either apoptotic or necrotic cell death, depending on the cell type and severity of insult. Further, effectuation of the apoptotic death program requires maintenance of a sufficient intracellular energy level and of a redox state compatible with caspase activity. Thus, ATP depletion or severe oxidative stress may redirect otherwise apoptotic cell death to necrosis.KeywordsApoptotic Cell DeathMitochondrial Permeability Transition PoreAdaptor MoleculeNecrotic Cell DeathThiol OxidationThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Biological reactive intermediates in drug discovery and development: a perspective from the pharmaceutical industry.

The detection of chemically-reactive, electrophilic metabolites poses a particular problem in the discovery and development of drug candidates in pharmaceutical research, inasmuch as it is not possible to accurately predict the likely toxicological consequences of these intermediates in animal safety studies or in clinical trials. Advances in analytical instrumentation (notably liquid chromatography-tandem mass spectrometry [LC-MS/MS]) have facilitated the detection of reactive intermediates through the identification of the glutathione (GSH) adducts to which they normally give rise, while the increased use of radiolabeled tracers in drug development permits an early assessment to be made of the propensity of a drug candidate to undergo covalent binding to cellular macromolecules. Unfortunately, these advances in analytical methods for the detection and characterization of reactive drug metabolites have far outstripped our understanding of the mechanisms of foreign compound-mediated toxicities at the molecular level, and of the role of both covalent binding and oxidative stress in the cascade of events that lead ultimately to cellular injury or immune-mediated toxicities. In light of these uncertainties, it seems reasonable to argue that one should attempt to minimize, through structural modification, the extent to which a drug candidate undergoes metabolism to reactive intermediates. Therefore, it becomes imperative to have close collaboration between Drug Metabolism scientists and their counterparts in Medicinal Chemistry during both the discovery and early development phases.

Biological reactive intermediates that mediate chromium (VI) toxicity.

1. Addition of Cr VI (dichromate) to isolated rat hepatocytes results in rapid glutathione oxidation, reactive oxygen species (ROS) formation, lipid peroxidation, decreased mitochondrial membrane potential and lysosomal membrane rupture before hepatocyte lysis occurred. 2. Cytotoxicity was prevented by "ROS" scavengers, antioxidants, and glutamine (ATP generator). Hepatocyte dichlorofluorescin oxidation (to determine ROS/Cr V formation) was inhibited by mannitol (a hydroxyl radical scavenger) or butylated hydroxyanisole and butylated hydroxytoluene (antioxidants). 3. The Cr VI reductive mechanism required for toxicity are not known. Cytotoxicity was also prevented by cytochrome P450 inhibitors, particularly CYP 2E1 inhibitors, but not inhibitors of DT diaphorase or glutathione reductase. This suggests that P450 reductase and/or reduced cytochrome P450 contributes to Cr VI reduction to Cr IV. 4. Glutathione depleted hepatocytes were resistant to Cr (VI) toxicity and much less dichlorofluorescin oxidation occurred. Reduction of dichromate by glutathione or cysteine in vitro was also accompanied by oxygen uptake and was inhibited by Mn II (a Cr IV reductant ). Cr VI induced cytotoxicity and ROS formation was also inhibited by Mn II which suggests that Cr IV and Cr IV.GSH mediate "ROS" formation in isolated hepatocytes. 5. In conclusion Cr VI cytotoxicity is associated with mitochondrial/lysosomal toxicity by the biological reactive intermediates Cr IV and "ROS".

Metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in A/J mouse lung and effect of cigarette smoke exposure on in vivo metabolism to biological reactive intermediates.

It has been proposed that the tobacco-specific N-nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) may be involved in the causation of human lung cancer (Hecht and Hoffmann, 1988), but direct evidence for the involvement of NNK in human lung cancer is lacking (Hecht and Tricker, 1999). In the A/J mouse lung tumor model, a single intraperitoneal (i.p.) injection of 10 μmole [2.07 mg] NNK/mouse results in 7–12 lung tumors per mouse after 16 weeks (Hecht et al., 1989). The initial events in NNK-induced A/J mouse lung tumorigenesis are believed to be metabolism of NNK to biological reactive intermediates (BRI) with the potential to react (via methylation of DNA) to produce O6-methylguanine (O6MeG), GC→AT transitional mispairing, and subsequent activation of the K-ras proto-oncogene (Ronai et al., 1993). α-Hydroxylation of the methylene carbon atoms adjacent to the N-nitroso group in NNK and NNAL yields unstable BM which spontaneously decompose to methanediazohydroxide with the potential to react with DNA to produce 7-methylguanine (7-MeG), O4-methylthymidine (O4MeT) and O6MeG adducts. α-Hydroxylation of the NNK methyl group yields a BM with the potential to pyridyloxobutylate DNA, while α-methyl hydroxylation of NNAL is not known to result in DNA adduct formation. Other metabolic transformations represent detoxification pathways for NNK and NNAL.KeywordsEnvironmental Tobacco SmokeHuman Lung CancerMouse LungCigarette Smoke ExposureLung TumorigenesisThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Sequestration of biological reactive intermediates by trapping as covalent enzyme-intermediate complex.

One important class of biological reactive intermediates arising in the course of human xenobiotic metabolism are arene and alkene oxides. The major safeguard against the potential genotoxic effects of these compounds is the microsomal epoxide hydrolase (mEH). This enzyme has a broad substrate specificity but--on the first sight--seems to be inadequately suited for this protection task due to its low turnover number with most of its substrates. The recent progress in the understanding of the mechanism of enzymatic epoxide hydrolysis has shed new light on this apparent dilemma: Epoxide hydrolases convert their substrates via the intermediate formation of a covalent enzyme-substrate complex, and it has been shown that the formation of the intermediate proceeds by orders of magnitudes faster than the subsequent hydrolysis, i.e. the formation of the terminal product. Thus, the enzyme acts like a molecular sponge by binding and inactivating the dangerous metabolite very fast while the subsequent product release is considerably slower, and quantification of the latter heavily underestimates the speed of detoxification. Usually, the slow enzyme regeneration does not pose a problem, since the mEH is highly abundant in human liver, the organ with the highest capacity to metabolically generate epoxides. Computer simulation provides evidence that the high amount of mEH enzyme is crucial for the control of the steady-state level of a substrate epoxide and can keep it extremely low. Once the mEH is titrated out under conditions of extraordinarily high epoxide concentration, the epoxide steady-state level steeply rises, leading to a sudden burst of the genotoxic effect. This prediction of the computer simulation is in perfect agreement with our experimental work. V79 Chinese Hamster cells that we have genetically engineered to express human mEH at about the same level as that observed in human liver are well protected from any measurable genotoxic effect of the model compound styrene oxide (STO) up to an apparent threshold level of 100 microM in the cell culture medium. In V79 cells that do not express mEH, STO triggers the formation of DNA strand breaks in a dose-dependent manner with no apparent threshold. Above 100 microM, the genotoxic effect of STO in the mEH-expressing cell line parallels the one in the parental cell line.

Chromosome damage from biological reactive intermediates of benzene and 1,3-butadiene in leukemia.

The causes of leukemia remain largely unknown. Only ionizing radiation, benzene and various chemotherapy drugs are established causes in humans. While benzene is an established cause of acute myeloid leukemias, its ability to cause lymphocytic leukemias and the closely related lymphomas and multiple myeloma remains highly controversial (1-4). The concentration at which it significantly increases leukemia risk is also contentious. For example, it was recently claimed in a leading journal that benzene has a dose threshold of 200 p.p.m.-years for leukemia induction (4), although there is clearly information that benzene induces leukemia and other hematological changes at much lower doses than this (3,5) and that its effects are likely to be linear down to background levels (6). Other occupational and environmental chemicals are suspected leukemogens, but are not established as such and controversy surrounds their classification as human carcinogens. One such compound is 1,3-butadiene. This compound is widely used in the rubber industry and is a product of incomplete combustion. Its’ potential classification as a human carcinogen on the basis of epidemiological studies (7,8) showing higher rates of leukemia in exposed workers is under consideration by numerous agencies (9). To throw further light on these important questions concerning the human risk posed by benzene and butadiene, we have examined the ability of benzene, butadiene and their reactive intermediates to produce the types of chromosome damage thought to be etiologically important in leukemia and lymphoma in exposed humans and in human cells in vitro. This paper summarizes our recent findings and discusses their impact on the risk assessments for benzene and butadiene.

Modelling the responses to biological reactive intermediates: establishing the borderlines of risk.

Biotransformation enzymes modify xenobiotic compounds into more hydrophilic products (metabolites) which can be efficiently excreted from the body. Phase 1 enzymes, of which the cytochromes P450 are the most important, perform functionalization reactions creating a reactive centre in the molecule through oxidation or reduction. Phase 2 enzymes, such as glutathione S-transferases, UDP-glucuronyltransferases, N-acetyltransferases and sulphotransferases couple an endogenous molecule to a functional moiety in the molecule or metabolite from the phase 1 reaction, generally leading to more hydrophilic metabolites that are easily excreted with urine or bile.

Use of covalent binding in risk assessment.

Risk characterization comprises hazard identification describing the intrinsic toxic potential of a chemical, toxicokinetics, as well as the toxic mechanisms, information about dose response and exposure assessment. Compounds that induce reversible effects, which are repaired during and after exposure, are considered thresholded and allow definition of a NOEL. If damage is not repaired, the effect persists and accumulates upon repeated exposure. In such cases a NOEL cannot be determined. Biological reactive intermediates of chemicals have the potential to bind covalently to cellular macromolecules like proteins and DNA. Such interaction is not repaired completely and may persist. Thus, data on covalent binding (CB) are of qualitative and quantitative significance in the risk assessment process. Qualitatively, CB, especially with DNA and in correlation with this to proteins, is indicative for an irreversible and non-thresholded mutagenic and carcinogenic effect. Absence or presence of CB assists to differentiate between primarily genotoxic and thresholded non-genotoxic carcinogens. Quantitatively, CB is used to understand internal exposure and target dose, which is a prerequisite for species-species extrapolation, and to justify extrapolation from high dose to low dose. The reactive intermediates of ethylene, propylene and styrene have been determined in rodents and humans and modeled to predict dose responses of internal exposure. It is described in this communication that such information, together with other parameters like cell proliferation as a result of cytotoxicity, is the basis for quantitative risk assessment of human exposure to these compounds.

Chemoprotection and interindividual differences in response to biological reactive intermediates.

In humans and experimental animals, carcinogenesis is a complex process in which normal cell growth is modified. Carcinogenesis is divided into three main stages: initiation, promotion and progression. Chemopreventive interaction in carcinogenesis offers two major strategies. The first strategy will inhibit or at least slow down carcinogenesis by blocking its progress. This might occurr at all stages of carcinogenesis. This strategy includes the scavenging of bioreactive intermediates (BRIs), induction or inhibition of enzymes of the metabolism of xenobiotics which create or detoxify the BRIs, of enzymes of DNA-repair or of other enzymes. A second strategy aims at reversing the process of tumour formation either by redifferentiation of transformed cells or the elimination of precarcinogenic clones. Both strategies cover one or several steps of carcinogenesis and approach the subject matter in more general terms without consideration of individual susceptibility to particular cancers (Kelloff et al., 1999).

An Unusual Dehalogenating Peroxidase from the Marine Terebellid Polychaete Amphitrite ornata

The terebellid polychaete Amphitrite ornata produces no detectable volatile halogenated secondary metabolites, but frequently inhabits coastal marine sediments heavily contaminated with anthropogenic or biogenic haloaromatic compounds. This animal contains high levels of two very unusual enzymes, dehalogenating peroxidases. We have purified and partially characterized one of these dehaloperoxidases, DHP I. DHP I is a heme enzyme (Mr = 30,790) composed of two identical subunits (Mr = 15,529) and is very rich in the amino acids aspartic acid (+ asparagine) and glutamic acid (+ glutamine). The enzyme converts trihalogenated phenols, such as 2,4,6-tribromophenol, into dihalogenated quinones. The optimum pH for this reaction is 5.0. DHP I is also active against di- and monohalogenated phenols and will oxidize bromo-, chloro-, and fluorophenols. We have identified similar dehaloperoxidase activities in other infaunal polychaetes, including halometabolite-producing species.