1. Hydrocarbons: Mechanisms of Cellular Toxicity

2. Organohalides Are Lipophilic Less available for excr’n Accumulate in fatty tissues, fat stores –Fatty tissue ½ life may be > 100 d –Secr’d in milk Bioaccumulate in aquatic spp –Partition out of water –  Sediments –  Fish fatty tissue

3. Organisms may accumulate 3-6x (or more) amt in env water www.ecoinfo.org/.../gbhtoxin/gbhtoxin_e.cfm

4. Polynuclear Aromatic Hydrocarbons Benzo(a)pyrene, benz(a)anthracene models Metabolized by mixed function oxidases –Cytochromes P450  Reactive diol epoxides Ox’d forms in bay region most toxic

5. bay region

6. Halogenated Hydrocarbons May Increase Their Own Toxicity Work through Ah receptor Induce prod’n Cyt P450 enz’s –  Oxidized cmpds –Ex: 7,8- Dihydro-7,8- dihydroxy- benzopyrene 9,10 epoxide

7. The Ah–receptor binds four classes of substances: Dibenzodioxines (i.e TCDD) A, Dibenzofuranes, B Biphenyls C and polyaromatic hydrocarbons D. If such substances reach the receptor E, they trigger a chemical signal, which will finally result in toxic phenomena: tumor growth, skin toxicity (i.e. Ah mediated chloracne) F, immunotoxicity as well as developmental toxicity. The dioxin receptor belongs to the class of receptors mediating toxicity, which are preferentially modelled by QSAR methods. 3r-training.tierversuch.ch/.../dioxin.html

8. Hydrocarbon binds Ah receptor in cytosol Complex translocates  nucleus  Specific recognition sites on DNA for complex –Ah gene locus Now transcr’n, transl’n initiated –Specific genes code for AHH Aryl hydrocarbon hydroxylase –Phase I enzyme –Structural gene for cytosolic receptor

10. Now more metabolic (Cyt P450) enz’s Catalyze more ox’n of parent cmpd Product now more toxic –Incr’d carcinogenesis May covalently bind DNA  Mutations, incr’d repl’n Increased Toxicity of Metabolites

12. PolyChlorinated Biphenyls “PCB’s” 210 poss congeners –Chem cmpd closely related to another in composition –Exerting similar or antagonistic effects –Something derived from same source or stock Widespread, persistent –In fat tissue of most humans

13. Two most potent –3,4,3’,4’-tetrachloro biphenyl –3,4,5,3’,4’,5’-hexachloro biphenyl

14. Halogen Substitutions Cl is a halogen –F, Cl, Br, I –Reactive –Wants 1 e- to fill outer shell Form ions w/ single negative charge

15. In halogenated hydrocarbons, Cl covalently bound to C –Electronically stable bond Most mol’s containing Cl man-made –  incr’d molecular stability, incr’d MW, incr’d bpt/mpt

16. Cl substitutions on biphenyl often balanced When ortho substituted w/ Cl –Cl relatively large –2 planar rings can’t rotate Rotation hindered by Cl’s –When no Cl, 2 planar rings can rotate About C1, C1’ bond

17. PCB Toxicity Low acute toxicity Chronic exposure not understood –Probably more harmful Combines w/ receptor –Ah (aryl hydrocarbon) receptor –PCB  PCB-Receptor  Nucleus  DNA  alter transcription  effect on cell

18. Three Modes of Action Bind cell macromol’s –DNA –“Stacks and sticks” to proteins Accumulate in lipid-rich cell components Reversibly bind receptors, enzymes –At specific sites –Ah receptor has great affinity for TCDD, PCB’s Via Cl’s

19. PCB Biotransformation Bioactivation depends on planarity More toxic (and more similar to dioxin) when –Coplanarity of rings –Cl’s at m, p positions Metab’d through Phase I and Phase II

20. Phase I Metab Cytochrome P450 monooxygenase common –Indirect hydroxylation OH added, then db in ring shifts –Epoxidation --O– added over db in ring –May or may not shift or lose Cl –Metab rate depends on #, placement Cl’s More rapid if >4 Cl’s and H’s on C’s 4,5

21. Phase II Metab Phase II Reactions –Conjugated to glucuronic acid Rapidly excr’d –Conjugated to GSH  Mercapturic acid Excr’d or reabs’d

22. Common Effects of PCB’s Note: many effects are species-specific Coplanarity of rings, #Cl’s related to potency Chloracne –Acneiform eruption w/ exposure –Milder than w/ TCDD Probably diff mech

23. Epithelial cell changes –Hyperplasia – Incr’d cell # w/ incr’d cell div’n –Hypoplasia – Decr’d cell division  decr’d # proliferating cells –Impt to changes in sev organs Hepatomegaly Gastric mucosal changes –  Ulceration, hemorrhage –Species specific –May play role in carcinogenesis Hyperplasia = incr’d cell division; may be precursor for … Cancer = unrestrained cell division

24. Vit A depletion –May be linked to Ah receptor Heme prod’n inhib’d –Get build-up of porphyrins which are toxic Immunosuppression –Lymph glands Spleen enlargement Thymus gland atrophy –Total serum Ig’s decline (species specific) Humans: IgA, IgM sig depressed Phagocyte #, T cell response depressed From Japan, Taiwan PCB poisoning epidemics

25. Nervous system disorders –Catecholamine levels changed –Behavioral, learning dysfunctions Offspring following prenatal exposure Endocrine disruption –Next week’s lecture

26. Dioxins Also widespread Also hydrophobic Most toxic –2,3,7,8- tetrachlorodibenzo- p-dioxin

27. “Supertoxic” cmpd –Extremely potent –Diff isomers differ in toxicity –Contaminant of other chlorinated cmpds Induces microsomal enz’s –May be through Ah receptor

28. TCDD binds Ah receptor in cytosol –Some spp (even diff strains) have varied amts Ah receptor in cytosol –More receptor/higher sensitivity to TCDD Complex translocates  nucleus  Specific recognition sites on DNA for complex –Ah gene locus

29. Now transcr’n, transl’n initiated –Specific genes code for AHH Aryl hydrocarbon hydroxylase –Phase I enzyme –Structural gene for cytosolic (Ah) receptor –Other prot’s impt for immune, inflamm responses (?) –Other prot’s impt to cell viability, replication

30. http://www.med.ufl.edu/pharm/facdata/Shiveric/images/TCDD.gif

31. Toxicity in Humans Uncertain Incr’d cancer mortality? –Carcinogenic in rats (@ 2 ppb)  Cancers of liver, resp tract, mouth, others –“Not reported to be carcinogenic in humans in spite of its extremely potent carcinogenicity in rats” (WHO, 1989) –German pesticide workers suffer 39% higher cancer mortality rate compared to other Germans. (Hanson, David; 1991)

32. Chloracne –Most characteristic lesion in humans –Related to Ah receptor Hepatotoxicity at high doses –See hyperplasia, enzyme induction, others Immunosuppression –T cells most likely target –Species specific –Reversible Teratogenicity/fetotoxicity –Cleft palate/Ah receptor linked in mice? –Prenatal exposure  biochem & behavioral changes in offspring

33. Some Problems in Assessing Toxicity TCDD, congeners may be by-products of manufacture along with PCB’s –So which is responsible for toxicity? DDT often present in mixtures, animals, environment –Pesticide, but also manufacturing by- product –OR may be in environment previously –OR may have been in animal fat from earlier exposure

34. One toxicant may influence others in mixture –Synergism or antagonism Which in a mixture is most toxic? –Can’t fully assess mechanisms Difficult to assess environmental risk –Molecule in largest concent may not be most toxic If one toxicant induces metab of another: ???? Species specificity impt –Fat stores? (ex: mink)