C.R. Acad. Sci.

Paris, Sciences de la vie / Life Sciences 324 (2001) 97106 2001 Acadmie des sciences/ditions scientiques et mdicales Elsevier SAS. Tous droits rservs S0764446900012816/FLA

Concise review paper / Point sur

Chemo-defence system
Andr Rico*
24, rue Balard, 75015 Paris, France
Received 22 May 2000; accepted 16 October 2000
Communicated by Roger Monier

Abstract By analogy with the immune defence system, the existence is suggested of a chemo-defence system protecting living organisms against toxic substances, whether natural or man-made, that are present in the environment. This paper deals rst with the various facets of such a system: mechanisms involving, among others, lipophilic compounds, hydrophilic compounds, oxidants, acidosis, genotoxics and metals; second, with the biological characteristics of the system and a comparison with the immune defence system : partial immaturity of the young, inducibility, non-specicity and specicity, and saturability; we will show that the two systems share many common features; third, with the evolution of the system, which demonstrates that the system is very old and suggesting that it came into existence before the immune defence system; and fourth, with some of its consequences: estimation of the toxic effects of low doses, hormesis, impact of a vegetable diet on health. Finally, it could be emphasised that life is well protected against chemicals by its chemo-defence system, which appeared very early with the rst living organisms on the earth. 2001 Acadmie des sciences/ditions scientiques et mdicales Elsevier SAS
chemo-defence / immune defence / biotransformations / low doses / hormesis / fruit and vegetable diet

Rsum Systme de chimio-dfense. Par analogie avec le systme immunitaire, on peut estimer quil existe un systme de chimio-dfense protgeant les tres vivants des innombrables toxiques chimiques naturels ou de synthse prsents dans leur environnement. Dans une premire partie, sont discuts les principaux mcanismes de ce systme, prsents suivant certaines caractristiques physico-chimiques ou biologiques des toxiques : composs lipophiles, composs hydrophiles, oxydants, acidiants, mutagnes, mtaux. Dans une deuxime partie, sont dcrites les caractristiques biologiques du systme : partielle immaturit chez les jeunes et inductibilit, non-spcicit et spcicit, saturabilit. Une comparaison est faite avec le systme immunitaire montrant que ces deux systmes sont de fait biologiquement assez proches. Dans une troisime partie est aborde lvolution du systme dmontrant aussi sa ralit. La dernire partie traite de quelques consquences de lexistence de ce systme : effets des faibles doses , concept dhormesis et hypothse daction protectrice dun rgime vgtarien, en particulier sur le dveloppement des cancers. Finalement, il apparat que la vie est bien protge des toxiques chimiques par son systme de chimio-dfense apparu trs tt sur terre avec les premiers organismes vivants. 2001 Acadmie des sciences/ditions scientiques et mdicales Elsevier SAS
chimio-dfense / systme immunitaire / biotransformations / faibles doses / hormesis / rgimes vgtariens

* Correspondence and reprints. E-mail address: andrerico@fr.europost.org (A. Rico).

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Version abrge
La vie sest dveloppe depuis son origine dans un environnement chimiquement et physiquement hostile : agents physiques (rayons UV solaires, rayonnements ionisants), composs chimiques prsents dans le milieu (mtaux lourds, oxygne) ou synthtiss par dautres organismes vivants pour agresser les proies ou se dfendre contre les prdateurs. Il est donc normal quau cours de lvolution soient apparus des mcanismes de dfense. Lobjet de cette note est dindiquer les analogies qui existent entre le systme immunitaire qui nous protge des agressions des organismes pathognes et certains mcanismes de dfense contre les toxiques chimiques qui peuvent tre considr comme partie intgrante dun systme homologue. Dans cette note, nous illustrerons ce concept par quelques exemples. Nous proposerons une classication des mcanismes en cause, nous soulignerons les analogies existantes entre systme immunitaire et systme de chimio-dfense et nous montrerons que la prsence de ces mcanismes inrme la navet de lhypothse selon laquelle toute quantit, si faible soit-elle, de toxiques chimiques fait courir un risque lorganisme vivant, ce qui amnerait la conclusion quil faut faire entrer les tres vivants dans des cocons protecteurs les mettant labri de tous toxiques physiques ou chimiques. Lintroduction de ce concept amne, au contraire, se demander si, comme pour le systme immunitaire, la prsence de petites quantits de produits toxiques nest pas ncessaire pour assurer le fonctionnement correct du systme chimio-dfense grce des stimulations priodiques et pour assurer son adaptation au milieu. Les produits chimiques peuvent tre classs en fonction de leurs proprits physiques, chimiques ou biologiques. Les composs dits lipophiles ont les plus grandes potentialits toxiques. Leur lipophilie leur permet de traverser aisment les membranes, donc dtre facilement absorbs par lintestin, la peau ou le poumon, sils sont volatiles. Ils pntrent aussi aisment dans les cellules et sils ne sont pas biotransforms ils saccumulent dans les graisses, voire dans le cerveau. Il existe heureusement des processus de biotransformations impliquant en particulier les cytochromes P 450, qui agissent sur leur structure pour les rendre plus polaires et faciliter leur limination par la bile et lurine. ces biotransformations, il faut ajouter les transporteurs multidrogues (MDT) existant dans toutes les membranes des cellules des tres vivants et plus particulirement exprims dans les pithliums, structures tissulaires en contact avec les substances chimiques lors de leur absorption (tractus digestif) ou de leur excrtion (rein). Les MDT sont non spciques et

expliquent la rsistance exceptionnelle aux antibiotiques de certains microorganismes tels que Pseudomonas aerginosa posant un rel problme de sant publique. Les composs hydrophiles sont nettement moins biodisponibles que les prcdents, mais certains sont toxiques aprs injection. Cest le cas, par exemple, des curares. Ces composs hydrophiles subissent aussi des processus de biotranformations non-cytochrome P 450 dpendants, assurant aussi leur dtoxication et leur limination. Loxygne et les espces oxydantes associes, comme lion superoxyde ou leau oxygne, sont des toxiques. Les oxydations dgradent les substrats, augmentent lentropie du systme donc le dsordre. La respiration cellulaire arobie peut tre assimile un mcanisme de dtoxication des potentialits toxiques de loxygne avec rcupration deffets biologiques positifs (synthse ATP). Quant aux superoxydes dismutases ou catalases, elles permettent de lutter contre les hyper oxydants et compltent laction de la respiration cellulaire. La rgulation acido-basique du pH sanguin peut tre aussi considre comme un processus de dtoxication en particulier vis--vis des acides. Les gnotoxiques sont des composs dangereux capables daltrer lADN. Cependant, dans les cas des faibles doses, il existe des systmes de rparation efficaces et varis. Ils sont constitutifs et inductibles. Par contre, si la dose ou le dbit de dose est lev, la probabilit de rparation fautive saccrot rapidement. Les mtaux sont aussi dangereux, mais ils peuvent tre dtoxis par les organismes vivants par complexation avec en particulier les mtallothionines, protines soufres rencontres chez toutes les espces. Le systme de chimio-dfense prsente un certain nombre de caractristiques biologiques analogues celles du systme immunitaire. Le systme de chimiodfense est en partie immature chez les jeunes, il sexprime par adaptation des organismes dans leur milieu soit par expression gnique soit par induction. Une question qui se pose est de savoir si linduction doit tre considre, comme cest souvent le cas en toxicologie classique, comme un effet non dsir. Cet aspect est discut. Le systme de chimio-dfense comme le systme immunitaire comprend une composante non spcique et une composante scique. En particulier, une analogie et une comparaison des structures entre cytochromes P 450 et immunoglobulines peuvent tre faites, expliquant dans les deux cas la grande spcicit de substrats exprime par ces deux types de molcules. Enn le systme de chimio-dfense, comme le systme immunitaire, est saturable. Cest la dose qui fait le poison, cest le quantum bactrien ou viral qui peut

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dclencher la maladie. Les deux systmes prsentent aussi des dsordres, par exemple de type hrditaire. Ltude de lvolution de ce systme avec le temps est aborde. Elle est intressante en ce sens quelle dmontre sa ralit et certainement son antriorit au systme immunitaire. De lexistence de ce systme de chimio-dfense, on peut tirer diffrentes consquences. Trois dentre elles sont voques. a) Effets des faibles doses : il est clair que lvaluation toxicologique actuelle gnralement fonde sur lextrapolation linaire est discutable et conduit au nal des limites tolrables plus dordre analytique que toxicologique avec tous les inconvnients que cela entrane, en particulier une chimiophobie de la socit. b) Le concept dhormesis : il indique que les faibles doses peuvent avoir des effets bnques, ce qui peut tre considr comme un corollaire

de lexistence dun systme de chimio-dfense. c) Le rle anticancreux tabli des rgimes base de fruits et de lgumes : celui-ci est gnralement considr comme li lexistence dantioxydants en nombre important dans ces aliments. Cependant, pris isolment ces composs (-carotnes, vitamines E, vitamines C, etc.) ne manifestent pas exprimentalement de telles proprits. Une autre approche consisterait dire que cest la complexit des structures chimiques rencontres chez les vgtaux par rapport celles rencontres dans les produits animaux, qui, en stimulant notre systme de chimio-dfense, nous protge efficacement. Au total, on peut donc dire que la vie est bien protge des substances chimiques par son systme de chimio-dfense apparu trs tt sur terre avec les premiers tres vivants.

1. Introduction
We are surrounded by bacteria, viruses, parasites and prions, but we survive, mainly because our body is protected by an immune defence system. We live in an ocean of chemical substances (carcinogens, mutagens, teratogens, endocrine disrupters, etc.). Most of these are natural xenobiotics which can be highly toxic (mycotoxines, alkaloids, heterosides, etc.). Man-made chemicals (pesticides, veterinary drugs, etc.) are also present in our environment. Sometimes in pathology problems arise from abnormal metabolites. Nevertheless, we survive. How? By analogy with protection against infectious diseases, we would like to suggest the existence of a system that includes a large number of mechanisms and that we could call the chemo-defence system. When living organisms appeared on the earth, they were confronted with a very hostile environment with a high ux of UV light and ionising radiation and many inorganic and some organic chemicals. In order to survive, the living organisms developed defence mechanisms such as DNA repair systems. When oxygen, which is toxic and was present at trace levels in the beginning of life, became more quantitatively important, some bacteria detoxied it by way of aerobic cellular respiration. The chemical medium became more complex with the plants. Thus, to face the many new organic chemicals, initial cytochrome P 450s, devoted at the beginning to metabolic pathways, became more diversied to detoxify complex liphophilic chemical structures. Subsequently, the multi-cellular organisms used all these chemo-defence mechanisms and organised them so as to survive. It is this complex organisation that could be dened as the chemo-defence system. I introduced this new concept very recently but only relatively briey [1]. In this basic conceptual research

paper, I would like to go into more scientic details to support my conceptual model. This paper will deal with the nature and function of the chemo-defence system, together with its biological characteristics, evolution and consequences. However, it will not cover all the toxic effects due to chemicals in detail. The same applies for references; it is not possible to cite all the articles devoted to the different sections. A choice has been made, but as in all choices, it could be open to criticism.

2. Nature and functions

Chemicals can be classied according to their physical properties, chemical properties or biological effects. We shall use this classication to discuss the major different categories of toxic products and the processes of detoxication associated with them.
2.1. Lipophilic compounds

In general lilophilic compounds present the greatest possibility of toxicity. Their lipophilic properties facilitate their penetration into the lipid membranes. This means that they are readily absorbed by the gut, they can go through the skin and, if they are volatile, they can rapidly penetrate the lung tissue. If they are not rapidly detoxied, some (e.g. organochloride insecticides such as DDT) may accumulate in fatty tissues, others (such as methyl selenium) can go through the bloodbrain barrier, often with highly toxic effects. They may also penetrate the cells. Their volumes of distribution, in terms of pharmacokinetics, are always high to very high.
2.1.1. Biotransformations

Biotransformation processes exist in some organs, particularly the liver, but also in the intestine, lung, kidney,

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skin and testes. These biotransformation processes are able to transform the structures of lipophilic xenobiotics, thereby increasing their polarity, facilitating their elimination by bile and urine and most often decreasing their toxicity [2, 3]. Classically there are two phases in this process. Phase I: this is a structural biotransformation involving an enzyme system referred to as the mixed function oxydase (MFO) system. In this system, cytochrome P 450 mono-oxygenases play an important role. They are constituted by a super family of more than 400 hemothiolateenzymes, with a great affinity for lipophilic substances. These enzymes permit the introduction of hydroxyl groups into the structure of lipophilic compounds, which increases their polarity and facilitates excretion. Phase II, or conjugated phase: the biotransformation products and others, if their structures are adequate, become conjugated with polar agents of conjugation, which increase their urinary and biliary excretions. This is the case with glucuronic acid, sulphates, etc. Almost all these enzymes are inducible. The cytochrome P 450s are very efficient and two remarks can be made about their action. In general, biotransformations are processes of detoxication. In some cases the action of cytochrome P 450s can lead to bioactivation and produce aggressive electrophilic metabolites [3]. These metabolites are attracted by the nucleophilic sites, present for example in proteins and DNA, and can bind covalently with these targets and often exhibit toxic effects. They are suspected of genotoxicity and carcinogenicity. However, at low dose levels, these electrophiles are trapped by the glutathion present in the cell cytoplasm. As for the residues of pesticides or veterinary drugs, the process of bioactivation leads to the formation of covalently bound residues which are considered not to be of toxicological signicance for consumers of food containing these residues [4]. Some cytochrome P 450s are not involved in detoxication but are essential to the synthesis of steroid hormones catalysing, for example, the aromatisation reaction that produces oestrogen in the female sex organs but also in the oxidative metabolism of fatty acids, prostaglandins, leukotrienes, biogenic amines and pheromones. To summarise, it would seem that the biotransformations of lipophilic chemicals by the MFO system globally represent much more a process of detoxication than toxication at least at low dose levels.
2.1.2. Multidrugtransporters (MDT)

secondary transporters, which mediate the extrusion of drugs from the cell in a coupled exchange with ions. P glycoproteins (Pgp) are plasma glycoproteins of about 170 kDa and belong to the superfamily of ATP-binding cassette transporters. Humans have two Pgp genes, MDR1 (class I) and MDR3 (also named MDR2, class II) located on chromosome 7. They are expressed to a greater of lesser extent in many normal tissues. High levels are found in tissues with absorption or excretory functions, especially in the apical plasma membrane of intestinal epithelial cells and the proximal tubules of the kidney. These could explain the resistance of cancers derived from epithelium cells to chemotherapy. On the contrary, the high sensitivity of CF-1 mice to the toxicity of avermectins, used as antiparasitic drugs and pesticides, is associated with Pgp deciency in the small intestine and the capillary endothelial cells of the bloodbrain barrier [6, 7]. Finally, the resistance of micro-organisms to antibiotics is in part related to the existence of MDT [8, 9]. It is the same for the chemo-resistance of cancer cells [10]. Such systems could be considered as an important part of the chemo-defence system [11] and are also usually directed more specically against lipophilic compounds that enter the cells [12].
2.2. Hydrophilic compounds

These compounds, because of their structure, show limited, if any, absorption by the gut or through the skin. If they penetrate the body after parenteral administration, for example, they do not penetrate the cells, which are protected by their lipophilic membranes. In terms of pharmacokinetics, their volumes of distribution are low to very low. They are rapidly excreted by the kidney and are not accumulated. However, they can be toxic in the body, often by acting on the cell membrane receptors as some analogous polar hormones (e.g. 2 agonists) [13]. Curares are good examples. Their oral bioavailability is very low, so they are not toxic by this route, but have a potent paralysing action on the muscles after injection. Streptomycin is also a highly polar compound and is not absorbed by the gut or active by oral route. These compounds are also subjected to biotransformations, not involving cytochrome P 450s, but other enzymes, such as epoxidehydrolase, esterase and amidase, or are conjugated. These processes decrease the toxicity of these compounds and facilitate their excretion. Some of them are also inducible.
2.3. Oxidants and oxidative species

In organisms ranging from bacteria to humans, process transmembrane transporters are present which confer resistance to toxic compounds [5]. On the basis of bioenergetic and structural criteria these transport systems can be divided into two categories: ATP-binding cassette (ABC) transporters, which use the release of phosphate bond-energy by ATP hydrolysis to pump drugs out of the cell;

Oxidations, or at least full oxidations, can be viewed as toxic aggressions. These reactions are exergonic reactions, which destroy substrates, increase the entropy of the system, and in other words introduce disorder. Substrate dehydrogenations are in fact also oxidation processes. Oxygen is interesting to consider. Nature has found an efficient process to combat its toxicity: the cellu-

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lar aerobic respiration that takes place in the mitochondria [14, 15]. The oxidative phosphorylation process consumes oxygen, reducing it in sequential steps to produce water and create adenosine triphosphate (ATP). This high-energy molecule is then used in the endergonic reactions involved in substrate synthesis. These endergonic reactions decrease the entropy and disorder of the system. Cellular aerobic respiration thus provides a perfect illustration of the potential of living organisms to transform what could be toxic effects into benecial effects. By-products of oxidation such as the oxidising radicals superoxide anion and H2O2 are also very aggressive, leading to what is known as oxidative stress [16]. However, several means of defence against oxidative stress exist which maintain such oxidants at a low steady state. The superoxide anion is eliminated in the mitochondria by a specic Mn-containing superoxide dismutase, similar to the bacterial forms. This enzyme is inducible. These enzymes also exist in the cytoplasm but they contain copper and zinc. H2O2 can be eliminated by glutathion peroxidase and catalase. Catalases are present in great quantities in the leukocytes and it should be noted that H2O2 is formed during inammation. These protective mechanisms are very important because it is now very well known that processes of oxidation are involved in many toxic or natural effects such as mutations, carcinogenesis, damage to lipids and degenerative diseases associated with ageing [17].
2.4. Acidosis

stressed, however, that such bioactivations are located in the endoplasmic reticulum, so that after formation these electrophiles have to go through the cytoplasm, penetrate the nuclear membrane, go through nucleoproteins, basic proteins with many nucleophilic sites, in order to reach and attack the DNA. On their way, the electrophiles encounter numerous nucleophilic sites present in the proteins or specic detoxifying substrates such as glutathion. In fact, we can say that the nucleus is the safe box of the cell, in which the most important molecule for its survival and future is DNA and that this molecule is very well protected [18, 19]. So if production of electrophilic compounds is certainly dose dependent, this is not the case for their mutagenic effects, because at low dose the ux of electrophiles created by bioactivation is too low and has little chance of modifying DNA. This strongly suggests a threshold for this kind of toxic metabolite. The existence of a threshold for mutagenicity and carcinogenicity has already been stressed by many authors. Several papers have challenged the validity of the linear no threshold relationship (LNT), concerning radiation [20, 21] and chemicals [22]. Even if a modication of DNA takes place it is now well established that numerous and various DNA repair systems exist that are able to correct errors of replication or external aggressions. They are very efficient, present in almost all tissues, including skin, and are also inducible [19].
2.6. Metals

In some diseases such as diabetes mellitus or in shock phenomena, a syndrome of acidosis occurs by the accumulation of acid metabolites, which can lead to death. This syndrome can be compared to a toxic effect. It is well known that the blood pH is maintained between 7.30 and 7.35. A decrease in the pH to 7.20 is lethal. Two mechanisms of regulation maintain this pH at the normal level. One is a physicochemical system constituted by the buffer systems in the plasma (carbonic acid/bicarbonate, etc.). This system becomes active immediately, but its effect is limited. The second is a physiological mechanism involved in the excretion of acids by the kidney, which eliminates protons, and the lung, which eliminates carbon dioxide. These two systems work synergically to efficiently regulate the blood pH and can be viewed as detoxifying mechanisms against acidic toxics.
2.5. Mutagens

Mutagenicity can be dened as an irreversible alteration of the DNA molecule which may, for example, lead to carcinogenicity. Many natural and synthetic mutagens exist in our environment. In toxicology one important question concerns the bioactivation process [2, 3]; bioactivations can lead to highly reactive electrophilic metabolites which are able to attack DNA. It should also be

Metals may be toxic [23]; however, it is important to stress that in fact the term toxicity in the case of a metal is not toxicologically correct. What is important as regards metal toxicity is what we term speciation (the chemical form of the metal) and the bioavailability (absorption after ingestion). These aspects are very well known, for example, for mercury, lead, chromium, arsenic, etc. The toxicity of metals is related to their ionic form; and such toxicity is lost when they are chelated or complexed. Substances able to chelate metals and thus to detoxify them exist in the body. This is the case of metallothioneins, sulphur proteins formed in water organisms, mammals and birds [24]. These molecules are able to chelate heavy metals and decrease their toxicity (mercury, cadmium, etc.). Ferritin and hemosiderin act in the same way for iron. The metallothioneins are inducible. In summary, numerous mechanisms exist that can detoxify the chemicals which penetrate the body. In fact, these mechanisms do not work separately but together and often synergistically. For example, when the MDT pump the toxic compounds in the blood outside of the cells, these compounds will be detoxied through biotransformations, specically in the liver and the kidney, and excreted. These mechanisms can be also complementary or redundant. They are complementary when during aerobic respiration some reactive oxygen species appear in mitochondria. These species are eliminated by the super-

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oxide dismutase enzyme. They are redundant in the protection of the genome: in the case of mutation, the various DNA repair systems act to restore the integrity of the molecule. However, other mechanisms participate in this restoration, such as the arrest of cellular division, to give time to the cells to repair the lesions before mitosis (control points in G2 and in phase M in the cellular cycle) [25, 26], or apoptosis, which eliminates the abnormal cells [27].

that the effect is not related to any specic structure [6]. This explains in part the difficulties of chemotherapists to ght against chemo-resistant cancer cells [31].
3.2.2. Specicity 3.2.2.1. Organ specicity

3. Biological characteristics
3.1. Immaturity and inducibility

It is well known that many biological differences exist between the infant, the young and the adult [28]. In terms of chemo-defence the very young are partially immature. The metabolism of xenobiotic compounds in the newborn is both qualitatively and quantitatively different from that in adults. The system changes progressively and adjusts following contact with toxic compounds of the environment. This can be considered as a consequence of the expression of existing genes and inducibility. Many of the enzymes participating in the chemodefence system are inducible. This is particularly the case of cytochrome P 450s, some conjugases, DNA repair enzymes, metallothioneins, etc. The question is whether induction has a benecial, adverse or toxic effect. In experimental toxicology, in long-term toxicology tests, this kind of biological effect is often associated with an increase in liver weight. Its signicance is a matter of judgement. It seems that when this effect is not associated with histopathological changes or any other biological effect, we can consider that it is an adaptive if not benecial effect, especially if we accept the fact that the cytochrome P 450s have specic substrates, as will be seen in the next chapter. Remark: These characteristics are very similar to those of the immune defence system which is also immature in the newborn and is also inducible. Even more so, the presence of antibodies, which are induced by pathogens, is not always a sign of illness but rather a sign of the bodys contact with the germ.
3.2. Non-specicity and specity

Not all organs or tissues are involved in the same way in the process of chemo-defence, especially in the case of biotransformations, which are pivotal in terms of detoxication. The liver is the main organ. All the cells in this organ are able to participate in the process, and are able to extract a considerable quantity of toxic agents and excrete them via the bile. The importance of this organ is not surprising, since a primary function of the liver is to receive and process chemicals absorbed from the gastrointestinal tract. But we also nd processes of detoxication to a lesser extent than in the liver in those organs involved in the absorption of toxic compounds (lung with clara cells, intestine, skin), in their elimination (kidney, lung), but also in the testes where there is continuous production of spermatozoa essential to species reproduction and survival. These organs constitute an organ defence system similar to that of the immune system.
3.2.2.2. Substrate specicity

The functions of the immune system are provided by two major mechanisms: a non-specic or constitutive mechanism not requiring prior contact with the inducing agent and lacking specicity, and a specic or adaptive mechanism directed against and specic for the eliciting agent [29, 30]. The same is true for the chemo-defence system.
3.2.1. Non-specicity

Multi-drug transporters are non-specic mechanisms. As it was stressed above, they exist more or less in all tissues and there are no really specic inhibitors showing

Many enzymes involved in detoxifying processes exhibit chemical function specicity but not substrate specicity. This is true, for example, of esterases, amidases, epoxihydrolases, etc. The original consensus was that the cytochrome P 450 systems were not highly specic to substrates, but were essential to transform lipophilic compounds. However, in the light of recent research, a brief discussion of this point may be of interest [32, 33]. It is becoming clear that all cytochrome P 450s possess a conserved structural core. This core contains the active site and heme pocket, and the parts of the protein involved in the integration of the enzyme into the phospholipid matrix of the endoplasmic reticulum. This environmental lipid matrix explains the affinity of the system for lipophilic compounds. However, although the structural core is apparently conserved among P 450s, there is sufficient variability between the protein sequences for it to be said that no cytochrome P 450s are structurally identical. This variability is without any doubt related to substrate specicity, which certainly exists, but the situation is so complex that up to now it has been very difficult to identify it clearly. However, in the future it should be possible to say: Tell me what you eat and I will tell you what specic cytochrome P 450s are in your liver. An interesting point is to compare the cytochrome P 450 structure with that of the immunoglobulin molecules [30]. An immunoglobulin consists of four peptide chains, two light chains and two heavy chains, held together by disulphide bonds. Furthermore, each heavy and light chain is subdivided into a variable and a constant region. It is the

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variable region that determines the molecular specicity for antigens while the constant region of the heavy chains is responsible for the biological activity of the molecule. The structure of cytochrome P 450s is similar to that of immunoglobulins, again supporting an homology between the immune defence system and the chemo-defence system.
3.3. Saturability

The chemo-defence system is saturable. In other words, when the quantity of toxic compounds becomes too high, the system is unable to control them, and toxic effects occur. This aspect is well illustrated by the basic toxicological principle: It is the dose, which makes the poison, stated rst by Paracelse and later emphasised by Claude Bernard But this is also true of the immune system. It is the bacteria or virus quantum that can launch the illness. The quantum is dependent on the nature of the agent and its pathogenicity in the same way as the dose in toxicology is a function of the nature of the toxic compound together with its potential toxicity. The targets for the immune system are living organisms, whereas for the chemodefence system they are chemical products. But in both cases, the aim is the same: to neutralise the undesirable elements, by killing them or changing them structurally to decrease their toxicity.
3.4. Dysfunction

Some dysfunctions of the system are due to the absence of some part of the process. For example, xeroderma pigmentosum is a hereditary disorder due to a deciency in some DNA repair mechanisms in the skin. This disorder causes an increased sensitivity to UV light, with a greatly increased incidence of skin cancer and premature death. The absence of certain enzymes can also explain the great sensitivity to certain compounds in different species. The cat is decient in sulfotransferase, so it is much more sensitive than other species to phenol compounds. Dysfunctions also exist in the immune defence system as hypersensitivity, allergy or autoimmunity. To summarise, the chemo-defence system presents biological characteristics that are similar from many points of view to the immune defence system: partial immaturity in the newborn, inducibility, non-specicity and specicity of organs and substrates, saturability and dysfunctions. These biological characteristics could support the existence of a chemo-defence system.

In the present review we describe the gene structure and evolution of the 10 known P450 gene families, eight of which exist in mammals. Unless otherwise indicated, percent similarity always refers to the comparison of full-length amino acid sequences derived in a few instances from protein sequencing but more than 90% of the time deduced from the cDNA nucleotide sequence. With these data, several conclusions about P450 gene evolution are apparent. 1. The P450 superfamily comprises at least 10 gene families. 2. The P450 superfamily is ancient and has expanded via divergent evolution. 3. The ancestral P450 gene, present probably more than 1.5 billion years ago, had a minimum of 22 exons. 4. Estimation of the unit evolutionary period (UEP; millions of years required for 1% divergence in amino acid sequence) may be difficult due to several instances of gene conversion between homologous P450 genes. 5. Compared with all of the microsomal P450 proteins, two mitochondrial P450 proteins, encoded by nuclear DNA, are more similar to the prokaryotic P450 protein. Most drugs and combustion products are derived from plants or are similar in chemical structure to plant metabolites (phytoalexins). It is proposed that four of these P450 gene families (1, 11, Ill and IV) have evolved and diverged in animals due to their exposure to plant metabolites and decayed plant products during the last one billion years. The overlapping substrate specicities of the P450 enzymes are remarkable. There are numerous examples of drugs and other foreign chemicals that are good substrates for the enzymes encoded by two or more P450 gene families gene families that have diverged so long ago as to be chromosomally non-linked.
4.2. Multidrug transporters (MDT)

4. Evolution
We emphasise that almost all of the mechanisms described in section 2 appeared with the rst living organisms on the earth.
4.1. Cytochrome P 450s

The structure, evolution and regulation of P450 genes are clearly described by Nebert and Gonzales [34]. They stated:

It is clear that the MDT appeared early in microorganisms living in hostile chemical environments. We reproduce here the concluding remarks of the paper cited in section 2.1.2 [5]. MDT are present in all living cells. This review shows that the function and structure of some multidrug transporters is conserved from bacteria to humans. The short generation time, and the ease of mutant selection and DNA manipulation associated with microorganisms offers great advantages over mammalian cells. Therefore, studies on multidrug transporters in microorganisms could bring valuable information about the general molecular mechanism and physiological role of these transport systems, and their implications in human disease. Much more, appreciation of the mechanism of such transporters is crucial for the development of effective new drugs able to suppress multidrug resistance in clinical settings. In a recent paper concerning the genome sequence of Pseudomonas aeruginosa [35], it was stated Pseudomonas aeruginosa is a ubiquitous environmental bacterium that is one of the top three causes of opportunistic human infections. A major factor in its prominence as a pathogen is its intrinsic resistance to antibiotics and disinfectants. Here we report the complete sequence of P.aeruginosa

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strain PA01. At 6.3 million base pairs, this is the largest bacterial genome sequenced, and the sequence provides insights into the basis of the versatility and intrinsic drug resistance of P. aeruginosa. Consistent with its larger genome size and environmental adaptability, P.aeruginosa contains the highest proportion of regulatory genes observed for a bacterial genome and a large number of genes involved in the catabolism, transport and efflux of organic compounds as well as four potential chemotaxis systems. We propose that the size and complexity of the P.aeruginosa genome reect an evolutionary adaptation permitting it to thrive in diverse environments and resist the effects of a variety of antimicrobial substances.
4.3. Oxidant and oxidative species

immune defence system, which came after the appearance of eukaryotic and multi-cellular organisms. Perhaps it is for this reason that the immune defence system seems more integrated and more complex than the chemodefence system. Consequently, it is clear that it is not cytochrome P 450s that are modelled on immunoglobulins but rather the contrary. Thus, this section on evolution supports and conrms the existence of a chemo-defence system.

5. Some consequences
5.1. Low doses/high doses

We indicated in section 2.3 that cellular aerobic respiration taking place in the mitochondria is an efficient process to combat the toxicity of oxygen. In the presence of oxygen, some micro-organisms developed cellular aerobic respiration. Mitochondria share many aspects with bacteria, so it was hypothesised that it was a transcript of a primitive bacteria. This bacteria was phagocyted by a larger cell and established an endosymbiosic relationship with the host. A recent paper [36] illustrates well this hypothesis: We conclude that MsFtsZ-mt is likely to have been acquired from an endosymbiotic -proteobacterium that was the ancestor of the present-day mitochondrion. We suggest that in the course of evolution. the gene was transferred from mitochondrion to nucleus and that the nuclear-encoded protein is now targeted back to the mitochondrion to play a role in the division of the organelle. This is the rst identication of a eukaryotic ftsZ whose protein seems to be specically targeted to the mitochondrion, and which may thus be related to the earliest mitochondrial division genes.
4.4. Mutagenicity and DNA repair

Life appeared and developed in a bath of ionising radiation, which was more intense a billion years ago than now. The micro-organisms living at that time were under this ux. Consequently many mutations of these microorganisms certainly occurred. But also at this time, bacteria developed DNA repair systems, which are found now from bacteria to humans. At a recent symposium [37], it was stressed that repair processes act according to a precise sequence of reactions in response to DNA lesions and such a programmed sequence is exquisitely regulated in bacteria. In humans, the example of xeroderma pigmentosum is a good illustration of the importance of the reparation systems in both bacteria and humans. To summarise, it must be stressed that the mechanisms protecting living organisms against chemicals are very old. They took place in the rst living organisms, bacteria, and were later transmitted to other ourishing organisms on the earth. This original chemo-defence system, which began with bacteria, was certainly developed before the

As the system is saturable, it functions correctly for low doses. It might be less effective for medium doses and even less so for high doses. Unfortunately in toxicological experiments, the effects of low doses are the black box of this area. Until now, it has been very difficult to explore the mechanism of the effects of low levels of toxic agents. Today, the estimation of risk for humans is generally based on the effects observed at medium doses and high doses. From these results, an extrapolation is made, taking into account what we call safety factors. This is a very conservative approach, often scientically false, which totally ignores the chemo-defence system. Now, with the application of the precautionary principle, we are moving more and more towards analytical tolerances, ignoring the toxicological signicance of these values, for synthetic chemicals such as pesticides, veterinary drugs and food contaminants. By doing this we are creating fear in the public, increasing chemo-phobia and spending a great deal of money for the control of problems that are far more a consequence of psychological poisons than real poisons. This was very recently the case of dioxins detected in chickens in France and Belgium, which had a considerable economic impact for nothing. Research efforts must be made in toxicology to explore the black box of low dose effects. We now dispose of valuable analytical techniques with limits of detection of ng or pg/kg and also of suitably adapted tools in molecular biology and other elds (e.g. gene identication and sequences, their expression, computed modellising structures, 2D electrophoresis, etc.) to be able to investigate in depth the mechanisms of action of toxic agents.
5.2. Hormesis concept

Hormesis can be dened as the adaptive effects of low levels of toxic agents. Although the situation was rst described for radiation [38, 39], and also for chemical agents [40, 41] recent papers are now demonstrating the validity of this concept [42] and its applications. However, until now, this new concept, which supports the fact that toxic agents that are detrimental to human and animal health above certain threshold levels may induce positive effects at a dose that is signicantly lower than the classical no effect level, has not been wholly accepted by a large part of the scientic community and the regulatory agencies.

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The existence of a chemo-defence system certainly provides a valid explanation of the hormesis concept. The induction of detoxifying enzymes and other proteins by low doses would support the data that imply a hormesis response. However, it is not surprising that the national authorities do not take this concept into account in assessing the risks, at least in terms of food contaminants. Risk assessments must determine whether and how to incorporate this information into public health decisions in a manner consistent with regulations, public safety and public trust. Nevertheless, in the future a change in policy will certainly take place to incorporate this new scientic concept [43].
5.3. Anti-carcinogenic effects of fruits and vegetables

Remark: This concept of the chemo-defence system could help change attitudes in our society, which currently considers chemicals to be poisonous, dangerous and destructive to humans and the environment [49].

6. Conclusion
It appears that a chemo-defence system exists in living organisms in the same way as the immune defence system. In terms of evolution, the chemo-defence system most likely came into existence before the latter. The chemodefence system seems to be the ancestor of the immune defence system, which was developed when multi-cellular organisms confronted the problem of pathogens. Thus, it is not surprising that the two systems have many common features and complementary roles to ght against chemicals and pathogens. The system can be stimulated by all the chemicals present in our environment, whether natural or synthetic. There is abundant proof that we can increase the performance of the muscles or cardio-vascular system by exercise and sport and of the brain by intellectual effort. It is even better known that a physiological or biochemical system, which is not stimulated, progressively loses its properties. So, why should it not be possible for an organism to stimulate its defence against the chemicals found in our environment? The aim of this paper is not to report original data, but to support, by the introduction of the chemo-defence system concept, the idea that living organisms are well armed to ght against chemicals, at least at low or medium doses. Certain details in this conceptual paper may be criticised and some basic aspects may be controversial. This is a good thing, because it will open up a new area of discussion and should initiate new scientic investigations: for example, into the role of the nucleus as the safe box of the cells in preventing aggression against DNA, on the substrate specicity of cytochrome P 450s, on the effects of low doses more specically on the validity of the LNT hypothesis, on the concept of hormesis and, last but not least, on the protective mechanisms of the effects of fruits and vegetables in our diet. Finally, it must be stressed that life is very well protected against chemicals by its chemo-defence system, which appeared very early with the rst living organisms on the earth.

Ames has reviewed the role of dietary factors that are anti-tumorigenic and thus included fruits and vegetables, folic acids, various antioxidants, some vitamins and traces of metals [44]. Recently, the following topics were discussed [45]: a) the anti-oestrogenic and anti-tumoric activity of diindolymethane present in cruciferous vegetables, b) the chemo-preventive action of organosulphur compounds in allium, c) the mechanism of action and chemopreventive activity of the isoavonoid genistein, and d) polyphenolic compounds in green tea. The protective effects of carotenoids [46], vitamins C and E, etc., as antioxidants have also been put forward. However, when the specic compounds suspected of being involved in anti-carcinogenic or protective effects are tested separately using classical toxicological tests, the results are more often negative. In the case of high doses of carotenoids, these compounds are even suspected of carcinogenicity [47]. So the available information suggests that the efficacy of fruits and vegetables in preventing cancer is not linked to one or other specic compound. In plants but not in animals, many chemical structures are suspected of toxicity. In terms of natural pesticides, for example, which are not the only chemical products in vegetables, it has been estimated that the human diet contains roughly 5 000 to 10 000 different natural pesticides and their breakdown products [48]. So why is a plant diet in fact effective against cancers? Probably because it brings together many different chemical structures that are more or less toxic. These complex chemical mixtures ingested at low doses may stimulate the chemo-defence system and enhance the resistance of the host.

Acknowledgements. I am extremely grateful to Professor M. Tubiana and Professor R. Monier for critically reviewing this manuscript and for their suggestions and advice.

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