Dioxin: Toxicity and Health Effects

Seveso disaster (12:37 pm July 10, 1976,) The accident occurred in a plant where 2,4,5 T (2,4,5 trichlorophenoxyacetic acid), a herbicide, was being produced from 1,2,4,5-tetrachlorobenzene by the nucleophilic aromatic substitution reaction with sodium hydroxide. An uncontrolled exothermic reaction during the manufacture of trichlorophenol produced the sudden release of a cloud of chemical . The presence of TCDD as the main component of the toxic cloud was made known 10 days after the accident. The Seveso disaster was so named because Seveso was the community most affected living 25 km north of Milan in the Lombardy region in Italy. Within days a total of 3300 animals were found dead, mostly poultry and rabbits. Emergency slaughtering commensed to prevent TCDD from entering the food chain, by 1978 over 80,000 animals had been slaughtered. children were quickly hospitalised with skin inflammation, nausea, headache, and eye irritation . By the end of August Zone A had been completely evacuated and fenced, 1600 people of all ages had been examined and 447 were found to suffer from skin lesions or chloracne.

What is Dioxin?

Dioxin is the generic name for a group of 210 organic compounds that have two linked benzene rings. These two benzene rings are linked with either two oxygen atoms (dibenzodioxins), or one oxygen atom (dibenzofurans). Although there are 210 individual chlorinated dioxin and furan molecules, only 17 compounds in this class7 dioxins and 10 furansare considered toxic The most toxic individual compounds are 2,3,7,8-tetrachlorodibenzodioxin and,2,3,7,8-pentachlorodibenzodioxin

Toxicity Assessment To estimate the total toxic activity of dioxins, furans, and PCBs, especially when found in mixtures, a weighted factor called the toxic equivalence quotient (TEQ) is used The toxicities of the 17 most dangerous compounds have been estimated. From these estimates, weighting factorscalled toxic equivalence factors (TEFs)have been calculated. To calculate the TEQ of a sample, concentrations of each compound must be measured and multiplied by each TEF. TEQ = (TEFi Conc. dioxini) + (TEFj Conc. furansj) + . . . The toxic equivalence quotient (TEQ) was first used in the United States by the New York Department of Health to estimate the toxicity of dioxin in a building fire.

Sources Not intentionally manufactured -- Sources may be grouped into four major types: Combustion and Incineration Sources: Incineration of wastes such as municipal solid waste, sewage sludge, hospital and hazardous wastes; metallurgical processes such as high-temperature steel production, smelting operations, and scrap metal recovery furnaces; and the burning of coal, wood, petroleum products, and used tires for power/energy generation.) Chemical Manufacturing/Processing Sources: As by-products from the manufacture of chlorine and such chlorinated compounds as chlorinated phenols (e.g., pentachlorophenol), PCBs, phenoxy herbicides (e.g., 2,4,5-T), chlorinated benzenes, chlorinated aliphatic compounds, chlorinated catalysts, and halogenated diphenyl ethers. Industrial/Municipal Processes: Chlorine bleaching processes in the manufacture of bleached pulp and paper, chlorination of naturally occurring phenolic compounds present in wood pulp Reservoir Sources: Dredging of soils, sediments, and organic matter

Fate of CDDs Emissions: may be transported in air long distances Persistent chemical compounds more halogenated: more stable Unbound forms may undergo photo-degradation. The ultimate environmental sink of these CDDs/CDFs is aquatic sediments. Because dioxins are fat-soluble, they tend to accumulate in the fatty tissue of animals, thus concentrate in the food-chain.

Routes of Human Exposure

95% diet (meat, dairy,fish, breast milk) Proximity to hazardous waste site or incinerator Air, water, skin contact: less significant Occupational exposure..

Toxico-kinetics Once absorbed into blood, 2,3,7,8-TCDD and related compounds readily distribute to all organs, the liver and adipose tissue being the primary disposition sites. Multiple studies suggest that distribution of this class of compounds to internal organs is dose dependent. At low doses in animal studies, adipose tissue serves as the major depot; at high doses, a major fraction is sequestered in the liver. 2,3,7,8-TCDD and related compounds metabolism is a detoxification process necessary for the biliary and urinary excretion of these compounds. Direct intestinal excretion of the parent compound is another route for excretion of 2,3,7,8-TCDD and related compounds that is not regulated by metabolism. Following absorption, a half-life for elimination was estimated to be 2,120 days (5.8Yr)

Dioxin Toxicity Potent toxicants having diverse biological responses in wide variety of organisms. Dioxins have a common toxic mechanism. Alter basic steps for cell growth and development. Broad impacts in animals including human beings include: Endocrine disrupting Immunological impacts Neurological impacts Skin Carcinogenic

Non Cancer Effects

Human Non-cancer Effects Chloracne : An acne-like eruption of blackheads, cysts, and pustules associated with over-exposure to certain halogenated aromatic compounds, such as chlorinated dioxins and dibenzofurans. The lesions are most frequently found on the cheeks, behind the ears, in the armpits and groin region. Persists 10 yearsViktor Yushchenko (Before and After dioxin poisoning )

Male Reproductive effects: Decreased testosterone, Reduced sperm count, Semen changes, Male feminization, Female Reproductive effects: Decreased fertility, Miscarriage, Endometriosis, Hormonal disbalance Foetal effects: Birth defects- Majority studied are maternal exposures(during Pregnancy), Decreased length (transient) & birth weight, Neurological and developmental problems Increased foetal death, preterm birth & neonatal death rate, Children Growth: The dioxins in a womans body are also passed to her nursing infant in her milk Alteration in reproductive system, delayed puberty, Tooth defects infants receiving mothers milk having dioxin

Other Effects:Hyper pigmentationimmune suppressionLiver enzyme changes Altered glucose response - Diabetes Altered fat metabolism- Blood lipids: Triglyceride/cholesterol Increased liver microsomal enzyme activityWasting syndromeLiver, spleen, thymus and bone marrow damage Nervous system damage Etc. ....... ........ ......

Effects on thyroid hormone The thyroid hormone plays a major role in the development of the brain. The predominant source of brain thyroid hormone in the fetus has been shown to be foetal serum thyroxine (T4). A decrease in T4 during brain development might bring about brain damage. Even low levels of dioxin or PCB exposure during the perinatal period can greatly influence neurological development by the effects of these chemicals on thyroid levels in the developing brain. Maternal and infants thyroid hormone status is related to perinatal PCB and dioxin exposure. Infants exposed to higher dioxin-TEQ levels have lower plasma T4 and higher TSH levels at 2 weeks after birth as shown by some of the studies.

Effects on the immune system Thymic atrophy is a common immunotoxic effect of dioxin in experimental animals. Perinatal exposure to TCDD leads to thymic atrophy and a suppression of cell-mediated immunity. Dioxins interfere with the differentiation of hematopoietic precursors in thymus. Gestational TCDD exposure also suppresses delayed-type-hypersensitivity (DTH) response Although the mechanism for TCDD-induced thymic atrophy has not been fully elucidated, involvement of the p27(Kip1) gene in the inhibition of thymocyte proliferation has been suggested

Human Cancer 2,3,7,8-TCDD, is a a Group 1 carcinogen, meaning a "known human carcinogen." TCDD increases the risk of cancer at multiple sites, including lungs carcinoma. Many studies have shown dioxins are related to increased incidence of breast cancer. Possible association with soft tissue sarcoma Little known if any association with Non Hodgkin Lymphoma or skin carcinoma There is no known "safe dose" or "threshold" below which dioxin will not cause cancer. TCDD might activate a gene(s) that is directly involved in tissue proliferation. TCDD-induced changes in hormone metabolism, which may lead to tissue proliferation

Protection Against Human Cancer

Although dioxin is clearly a potent tumor promoter, it may have a protective effect for breast cancer lifetime dietary administration of low doses of dioxin to rats resulted in a decrease in age-dependent spontaneous mammary and uterine tumors, as shown by some studies.

Transforming growth factor (TGF)Epidermal growth-factor (EGF) receptorsTumour necrosis factor (TNF )

Source- Environmental Health Perspectives, Vol. 102, Supplement 9: Toxicological Evaluation of Chemical Interactions (Nov., 1994), pp. 157-167

Biochemical Events of Toxicity TCDD acts via an intracellular protein (the Ah receptor), which is a ligand-dependent transcription factor that functions in partnership with a second protein (known as Arnt). Functional Ah receptors have been found in many human tissues, including lymphocytes, liver, lung, and placenta. The human population show polymorphism of Ah receptor structure and function. Therefore, humans may differ from one another in their susceptibilities to TCDD. The binding and hydrodynamic properties of the Ah receptor differ relatively little across species and tissues yet responses vary widely; it is impossible, therefore, to account for the diversity of TCDD's biological effects by characteristics of the receptor alone.

Dioxin as an Endocrine Disrupting Chemical

Estrogenicity of Dioxins

ubiquitin-ligase complex

Proteasome-mediated, dioxin-dependent degradation of ER and/or (AR)

AhR consists of HLH, PAS, acidic () and Q-rich structural domains. Binding of dioxins, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (D), to the AhR protein, leads to a conformational change that partitions AhR into two complexes. 1st:- A transcriptionally active complex is composed of AhR, ARNT, ER and a coactivator such as p300 interacts with the oestrogen-responsive element (ERE), causing the promoters of oestrogen-responsive genes to activate transcription in the absence of oestrogen. 2nd:- A ubiquitin-ligase complex-- Dioxins alter the abundance of steroid receptors with the help of the proteasome a large protein complex that recognizes proteins tagged with a chain of ubiquitin molecules and catalyses their degradatio. Dioxin promotes the formation of an atypical substrate-specific E3 ubiquitin ligases that involves a cascade of ubiquitin-transfer reactions culminating in the attachment of ubiquitin to the target protein ER or androgen receptors (AR)

Microbial Role in Dioxin Biodegradation

Pathway of 2-CDD biodegradation by aerobic bacteria.Compound definitions: 2-chlorodibenzo-p-dioxin (1);8-chloro-cis-1,10a-dihydroxy-1-hydro-dibenzo-p-dioxin (2); 4-chloro-2,2,3-trihydroxydiphenyl ether (3); 2-hydroxy-6-oxo-6-(4-chloro-2-hydroxyphenoxy)-2,4-hexadienoic acid (4); 4-chlorocatechol (5); 4-chloroguaiacol (6).

Pathway of 2-CDF biodegradation by aerobic bacteria. Compound definitions: 2-chlorodibenzofuran (1);8-chloro-cis-4,4a-dihydroxy-4-hydro-dibenzofuran (2); 5-chloro-2,2,3-trihydroxybiphenyl (3); 2-hydroxy-6-oxo-6-(5-chloro-2-hydroxyphenyl)-2,4-hexadienoic acid (4); 6-chloro-2-methyl-4H-chroman-4-one (5); 5-chlorosalicylic acid (6); 2-hydroxypenta-2,4-dienoate (7).

Proposed pathway of 2,7-dichlorodibenzo-p-dioxin (27-DCDD) degradation by the white-rot fungus, Phanerochaete chrysosporium . Legend: 27-DCDD (1); 4-chloro-1,2-benzoquinone (2), 4-chlorocatechol (3); 2-hydroxy-1,4-benzoquinone (4), 2-methoxy-1,4-benzoquinone (5); 4-chloroveratrole (6); 1,2,4-trihydroxybenzene (7); 2-methoxyhydroquinone (8); 4-hydoxy-1,2-benzoquinone (9); -ketoadipic acid (10);lignin peroxidase (LiP); manganese peroxidase (MnP)

Thus microbes are the only hope for the bioremediation of contaminated sitesPotential microbes: Sphingomonas, Pseudomonas, Burkholderia and Phanerochaete

Prevention

No treatment options are available Avoid contact with soils near contaminated or hazardous waste sites Discourage children from eating dirt/putting toys in their mouth, hand hygiene Diet Fish advisory Avoid food sources from contaminated areas Wash & peel fruits & vegetables from contaminated areas Smoking/alcohol Recreation Do not stop breast feeding

Bioassay/biomarker technology for screening of dioxins and dioxin-like compounds

The chemical methods used for analyzing dioxin-like compounds are primarily gas chromatography (GC) with mass spectroscopy (MS) electron capture detection (ECD). The biological methods in operation included biomarkers (wildlife/human effects), whole animal exposures (in vivo, laboratory exposure), cell- or organ-based bioassays (e.g., EROD, in vitro luciferase), and protein binding assays (e.g., ligand binding as well as immuno- assays)

EROD bioassay Currently, the EROD method is more commonly used, measuring the binding of the dioxin-like compound to the AhR and the subsequent induction of CYP1A related deethylation of 7-ethoxyresorufin to resorufin. In this bioassay, several CYP activities can be measured by using different substrates, e.g., for CYP1A1 (EROD), for CYP1A2 (methox-yresorufin- O -demethylase; MROD), and for CYP2B1(pentoxyresorufin-O-depenthylase; PROD). Several cell lines have been used for EROD bioassay. Order of responsiveness towards TCDD: Rat H4II > MCF -7 human breast cell lines> human Hep G2 > Hep 3 cell lines etc.

Bioluminescent Biosensors Based on Genetically Engineered Cells Genetically engineered cells used : bacteria, yeasts, or mammalian cells. Recombinant cell lines are prepared by stable transfection of wild type cells with reporter genes under transcriptional control of the DRE. The most common reporter genes are firefly luciferase (luc), bacterial luciferase (lux), green fluoroscent protein, galactosidase or others.

Green uorescent protein (GFP) provide superior spatial resolution of gene expression but less sensitivity. A biosensor based on bacterial luciferase can glow without adding a substrate. The lux CDABE gene cassette, encodes for both bacterial luciferase and the enzymes involved in the synthesis of its substrate. Firey luciferase, which emits yellow-green (557 nm) light, and the corresponding cDNA have been used successfully as a BL reporter of gene expression in bacterial or mammalian cells, but they require external addition of substrates.-galactosidase can be used as a reporter gene. The -gal activity can be detected using a chemiluminescent 1,2-dioxetane--D-galactopyranoside substrate.Bioluminescent Biosensors Based on Genetically Engineered Cells

Luciferase + ATP + Luciferin +O2 Light + AMP + Oxyluciferin + CO2 (Detection luminometer)EROD assay : 7 Ethoxyresorufin Cyt P450 1A1 Resorufin (Detection Flurometer)

Enzyme Immunoassays Dioxin specific antibodies are immobilized on EIA tube surface. Then sample is added that will bind anti dioxin antibodies. A competitor enzyme is then added which binds to the free sites of the antibodies. The amount of conjugate is inversely related to the amount of PCDD/PCDF bound on the EIA tube. A chromogenic substrate is added and colour intensity is determined using a spectrometer to measure the optical density which is proportional to the enzyme amount and inversely proportional to the PCDD/PCDF amount in the tube.

Conclusion Prevention is better than cure to meet with dioxin toxicity. Dose response relationship should be better studied in order to develop potent anticancer drugs Bioremediation aspects involving microbial metabolism should be given more emphasis for remediation of contaminated sites Effective biosensor technologies should developed for rapid and cost-effective screening of dioxin like compounds.

Most Welcome for Questions........

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