dioxins and dioxin-like compounds dioxins and dioxin-like compounds dioxins and dioxin-like compounds (dlcs) are a group of chemical compounds that are persistent organic pollutants (pops) in the environment. they are mostly by-products of burning or various industrial processes - or, in case of dioxin-like pcbs and pbbs, unwanted minor components of intentionally produced mixtures. some of them are highly toxic, but the toxicity among them varies 30,000-fold. they are grouped together because their mechanism of action is the same. they activate the aryl hydrocarbon receptor (ah receptor), albeit with very different binding affinities, leading to high differences in toxicity and other effects. they include: polychlorinated dibenzo-p-dioxins (pcdds), or simply dioxins. pcdds are derivatives of dibenzo-p-dioxin. there are 75 pcdd congeners, differing in the number and location of chlorine atoms, and 7 of them are specifically toxic, the most toxic being 2,3,7,8-tetrachlorodibenzodioxin (tcdd). polychlorinated dibenzofurans (pcdfs), or furans. pcdfs are derivatives of dibenzofuran. there are 135 isomers; 10 have dioxin-like properties. polychlorinated biphenyls (pcbs), derived from biphenyl, of which 12 are "dioxin-like". under certain conditions pcbs may form dibenzofurans through partial oxidation. polybrominated analogs of the above classes may have similar effects. "dioxin" can also refer to 1,4-dioxin or p-dioxin, the basic chemical unit of the more complex dioxins. this simple compound is not persistent and has no pcdd-like toxicity. polychlorinated dibenzo-p-dioxins polychlorinated dibenzofurans polychlorinated biphenyls 1,4-dioxin dioxins have different toxicity depending on the number and position of the chlorine atoms. because dioxins refer to such a broad class of compounds that vary widely in toxicity, the concept of toxic equivalency factor (tef) has been developed to facilitate risk assessment and regulatory control. tefs exist for seven congeners of dioxins, ten furans and twelve pcbs. the reference congener is the most toxic dioxin tcdd which per definition has a tef of one. in essence, multiplying the amount of a particular congener with its tef produces the amount toxicologically equivalent to tcdd, and after this conversion all dioxin-like congeners can be summed up, and the resulting toxicity equivalent quantity (teq) gives an approximation of toxicity of the mixture measured as tcdd. dioxins are virtually insoluble in water but have a relatively high solubility in lipids. therefore, they tend to associate with organic matter such as plankton, plant leaves, and animal fat. in addition, they tend to be adsorbed to inorganic particles, such as ash and soil. dioxins are extremely stable and consequently tend to accumulate in the food chain. they are eliminated very slowly in animals, e.g. tcdd has a half-life of 7 to 9 years in humans. incidents of contamination with pcbs are often reported as dioxin contamination incidents since these are of most public and regulatory concern. dioxins are a group of chemically related compounds that are persistent environmental pollutants. dioxins are found throughout the world in the environment, and they accumulate in the food chain, mainly in the fatty tissue of animals. dioxins are highly toxic and can cause reproductive and developmental problems, damage the nervous and immune systems, interfere with hormones, and also cause cancer. owing to the highly toxic potential of dioxins, efforts need to be undertaken to reduce current background exposure. in these regards, prevention or reduction of human exposure and a strict control of industrial processes to reduce formation of dioxins are warranted. chemistry there are 75 possible congeners of polychlorinated dibenzo-p-dioxins, but only 7 of them have affinity for the aryl hydrocarbon receptor (ah receptor) and are toxic via this mechanism. the crucial structures are so called lateral chlorines in positions 2,3,7, and 8. these 4 chlorines also make the congeners persistent, because they prevent microbial degradation. additional chlorines make the compounds less potent, but basically the effects remain the same although at higher doses. there are 135 possible dibenzofurans, and 10 in which the lateral chlorines are dioxin-like. critical structures of pcdd/fs. there are 209 pcb compounds. analogously to pcdds at least two lateral chlorines in each ring in positions 3,4, and/or 5 are needed for dioxin-like activity. because the ah receptor requires a planar (flat) structure, only pcb congeners that can rotate freely along the c—c axis between the rings can attach the receptor. substituents in ortho-positions 2 and 6 prevent rotation and thus hinder the molecule from assuming a planar position. mono-ortho congeners (one cl in 2, 2', 6, or 6') have minimal activity. no significant dioxin-like activities have been noticed, if there are two or more o-chlorines. brominated dioxins and biphenyls have similar properties, but they have been studied much less. structures of biphenyl and 3,3’,4,4’,5-pentachlorobiphenyl many natural compounds have very high affinity to ah receptors. these include indoles, flavones, benzoflavones, imidazoles and pyridines. these compounds are metabolized rapidly, but continuous intake from food may cause similar receptor activation as the background levels of dioxins. they do not reach concentrations causing typical dioxin-like toxicity, however. mechanism of action a schematic diagram of some ahr signaling pathways. the canonical pathway is depicted with solid black arrows, alternative pathways with dashed arrows, and an intersection of these two with a solid red arrow. the green bars represent the ahr, red bars arnt, yellow bars ara9 (aip, xap2), blue bars hsp90 and the blue ovals p23. dioxin binding to the ahr (1.) leads to its translocation into the nucleus by importin-β, (2.) heterodimerization with arnt and binding to the dna at dres, (3.) modulating expression levels of target genes (green arrows). one of the gene products elevated by this mechanism is ahrr, a repressor protein which forms a feedback loop that inhibits ahr action. the ahr is finally degraded by the ubiquitin–proteasome system (4.). ahr activation can also rapidly increase intracellular ca2+ concentration (5.) which in turn may ultimately result in augmented cox2 gene expression. elevation of ca2+ activates camks, which appear to have a critical role in the translocation of the ahr. another example of effects mediated by the ahr via non-canonical pathways is suppression of acute-phase proteins (6.) which does not involve dna binding. (simplified and modified from lindén et al.) the ah receptor is an ancient receptor, and its many functions have been revealed only recently. it is an over 600 million year old protein occurring in all vertebrates, and its homologs have been discovered in invertebrates and insects. it belongs to basic helix–loop–helix-pas proteins and acts as a transcription factor modifying transcription of a number of genes (see figure). ah receptor activity is necessary for normal development and many physiological functions. mice lacking the ah receptor (knockouts) are sick with cardiac hypertrophy, liver fibrosis, reproductive problems, and impaired immunology. the ah receptor is relevant in toxicology for two very different reasons. first, it induces several enzymes important in the metabolism of foreign substances, so called xenobiotics. these include both oxidative phase i enzymes and conjugative phase ii enzymes, e.g. cyp1a2, cyp1b1, cyp2s1, cyp2a5, aldh3, gsta1, ugt1a1, ugt1a6, ugt1a7 and nqo1. this is in essence a protective function preventing toxic or carcinogenic effects of xenobiotics, but in some conditions it may also result in the production of reactive metabolites that are mutagenic and carcinogenic. this enzyme induction can be initiated by many natural or synthetic compounds, e.g., carcinogenic polycyclic hydrocarbons such as benzo(a)pyrene, several natural compounds, and dioxins. secondly, ah receptors are involved in the activation or silencing of genes that lead to the toxic effects of high doses of dioxins. because tcdd at high doses can influence the transcription of perhaps hundreds of genes, the genes crucial for the multitude of toxic effects of dioxins are still not known very well. binding of dioxin-like compounds to the ah receptor has made it possible to measure total dioxin-like activity of a sample using calux (chemical activated luciferase gene expression) bioassay. the results have been comparable to teq levels measured by much more expensive gas chromatography-high resolution mass spectrometry in environmental samples. toxicity dioxin toxicity is based on inappropriate activation of a physiologically important receptor, and therefore dose-response must be carefully considered. inappropriate stimulation of many receptors leads to toxic outcomes, e.g. overdose of vitamin a leads to inappropriate activation of retinoid receptors resulting in e.g. malformations, and overdoses of corticosteroids or sex hormones lead to a multitude of adverse effects. therefore it is important to separate the effects of low doses causing activation of the receptor around the physiological range from the effects of high toxic doses. this is all the more important because of large differences in exposures even among human beings. western populations today are exposed to dioxins at doses leading to concentrations of 5 to 100 picograms/g (as teq in body fat), and the highest concentrations in accidental or deliberate poisonings have been 10,000 to 144,000 pg/g leading to dramatic but not lethal outcomes. the most relevant toxic outcomes of dioxins both in humans and animals are cancer and the developmental effects of offspring. both have been documented at high doses, most accurately in animal experiments. as to developmental effects there is an agreement that the present dioxin levels in many populations are not very far from those causing some effects, but there is not yet consensus on the safe level. as to cancer, there is a disagreement on how to extrapolate the risk from high toxic doses to the present low exposures. while the affinity of dioxins and related industrial toxicants to the ah receptor may not fully explain all their toxic effects including immunotoxicity, endocrine effects and tumor promotion, toxic responses appear to be typically dose-dependent within certain concentration ranges. a multiphasic dose–response relationship has also been reported, leading to uncertainty and debate about the true role of dioxins in cancer rates. the endocrine disrupting activity of dioxins is thought to occur as a down-stream function of ah receptor activation, with thyroid status in particular being a sensitive marker of exposure. tcdd, along with the other pcdds, pcdfs and dioxin-like coplanar pcbs are not direct agonists or antagonists of hormones, and are not active in assays which directly screen for these activities such as er-calux and ar-calux. these compounds have also not been shown to have any direct mutagenic or genotoxic activity. their main action in causing cancer is cancer promotion. a mixture of pcbs such as aroclor may contain pcb compounds which are known estrogen agonists but are not classified as dioxin-like in terms of toxicity. mutagenic effects have been established for some lower chlorinated chemicals such as 3-chlorodibenzofuran, which is neither persistent nor an ah receptor agonist. toxicity in animals high doses. the symptoms reported to be associated with dioxin toxicity in animal studies are incredibly wide-ranging, both in the scope of the biological systems affected and in the range of dosage needed to bring these about. acute effects of single high dose dioxin exposure include reduced feed intake and wasting syndrome, and typically a delayed death of the animal in 1 to 6 weeks. by far most toxicity studies have been performed using 2,3,7,8-tetrachlorodibenzo-p-dioxin. the ld50 of tcdd varies wildly between species and even strains of the same species, with the most notable disparity being between the seemingly similar species of hamster and guinea pig. the oral ld50 for guinea pigs is as low as 0.5 to 2 μg/kg body weight, whereas the oral ld50 for hamsters can be as high as 1 to 5 mg/kg body weight. even between different mouse or rat strains there may be tenfold to thousandfold differences in acute toxicity. many pathological findings are seen in the liver, thymus, and other organs. some effects such as thymic atrophy are common in many species, but e.g. liver toxicity is typical in rabbits. low doses. very few signs of toxicity are seen in adult animals after low doses, but developmental effects may occur at low dioxin levels, including foetal, neonatal, and possibly pubescent stages. well established developmental effects are cleft palate, hydronephrosis, disturbances in tooth development and sexual development, and endocrine effects. surprisingly, enzyme induction, several developmental effects and aversion to novel foods occur at similar dose levels in animals that respond differently to acute high-dose toxicity. therefore it has been suggested that dioxin effects be divided to type i effects (enzyme induction etc.) and type ii effects (lethality, liver damage, anorexia, and tumour promotion). the reason may be different requirements of the transactivation domain structure of the ah receptor for different genes. some of these low-dose