1. Synthesis, properties and health effects of oxygenated metabolites of PCBs Hans-Joachim Lehmler Department of Occupational and Environmental Health The University of Iowa

2. Oxygenated metabolites of PCBs: Synthesis, properties and health effects Hans-Joachim Lehmler Department of Occupational and Environmental Health The University of Iowa

3. Metabolism of Xenobiotics PHASE I METABOLISM Introduction or exposure to a functional group by oxidative or reductive processes or by hydrolysis (-OH, -NH 2, -SH, -COOH) e.g.: Cytochrome P450 (microsomes) PHASE II METABOLISM Reaction of a cofactor with an existing or created functional group e.g.: glucuronide conjugation Biological effects of metabolites Goal: to transform a lipophilic compound into a more hydrophylic species (metabolite) that can be excreted in urine or bile.

4. Overview PCBs as environmental contaminants PCBs as environmental contaminants Synthesis of hydroxylated PCBs Synthesis of hydroxylated PCBs Physicochemical properties Physicochemical properties PCB metabolism: arene-oxide, hydroxy and quinone metabolites PCB metabolism: arene-oxide, hydroxy and quinone metabolites Biological effects Biological effects

5. Polychlorinated Biphenyls (PCB) 209 congeners: 209 congeners: Complexe mixtures manufactured for use in transformers, capacitors, hydraulic fluids, etc. Complexe mixtures manufactured for use in transformers, capacitors, hydraulic fluids, etc. Production banned in 70s Production banned in 70s Lipophilic, bioaccumulate and bioconcentrate Lipophilic, bioaccumulate and bioconcentrate Found in environment and in animal and human tissues Found in environment and in animal and human tissues Cause adverse health effects in animals and in humans. Cause adverse health effects in animals and in humans.

6. Synthesis of Hydroxylated PCBs: Suzuki Coupling High yield synthesis Selective coupling Limitations: only 1 or 2 ortho-chlorine substituent From: Lehmler et al., Chemosphere 2001

7. Synthesis of Hydroxylated PCBs: Cadogan Coupling Low yield reaction Tedious purification Low selectivity (i. e. several products) Suitable for PCB and metabolite synthesis

8. Synthesis of Hydroxylated PCBs: Ullmann Coupling Suitable for PCBs with multiple ortho substituents Unsymmetric coupling: three products DISADVANTAGE: formation of toxic byproducts

9. 3D Structure of PCBs: Calculated Dihedral Angle (Calculated with MM2* using GB/SA water solvent continuum as implemented by MACROMODEL5.0)

10. Dihedral Angle in the Solid State (Crystal Structure) Calculated  experimental dihedral angle   Packaging problems in the solid state   Also: “packaging problems” in the receptor binding site   The biologically relevant dihedral angle of a PCB derivative is probably best described by a range of dihedral angles Calculated dihedral angle: 37.2° Solid state dihedral angle: 43.1° From: Lehmler et al., Acta Cryst. 2001

11. Chiral PCBs (metabolites): Example of Axial Chirality 19 PCBs form stable rotational isomers 19 PCBs form stable rotational isomers 7 chiral PCBs are of environmental concern 7 chiral PCBs are of environmental concern 3 or 4 ortho chlorine substituents are required to prevent racemization 3 or 4 ortho chlorine substituents are required to prevent racemization (  G = 177 (Cl 3 ) and 246 (Cl 4 ) kJ/mole) Also: Chiral hydroxy and methylsulfonyl metabolites Also: Chiral hydroxy and methylsulfonyl metabolites Lehmler et al., FEB 2003

12. Octanol-Water Partition Coefficient of Hydroxy PCBs Octanol-water partition coefficient: measure for the lipophilicity Octanol-water partition coefficient: measure for the lipophilicity PCBs and PCB metabolites: highly lipophilic PCBs and PCB metabolites: highly lipophilic Lipophilicity: Lipophilicity: Hydroxy PCBs < PCBs  PCBs bioaccumulate and biomagnify PCB Hydroxy PCB

13. Other Properties of PCBs PCBs… bioaccumulate and biomagnify bioaccumulate and biomagnify are semivolatile are semivolatile are poorly watersoluble are poorly watersoluble Vapor Pressure: Biomagnification Factor in Fish: Solubility in Water at 25°C:

14. Overview PCBs as environmental contaminants PCBs as environmental contaminants Synthesis of hydroxylated PCBs Synthesis of hydroxylated PCBs Physicochemical properties Physicochemical properties PCB metabolism: arene-oxide, hydroxy and quinone metabolites PCB metabolism: arene-oxide, hydroxy and quinone metabolites Biological effects Biological effects

15. Metabolism of Xenobiotics PHASE I METABOLISM Introduction or exposure to a functional group by oxidative or reductive processes or by hydrolysis (-OH, -NH 2, -SH, -COOH) e.g.: Cytochrome P450 (microsomes) PHASE II METABOLISM Reaction of a cofactor with an existing or created functional group e.g.: glucuronide conjugation Biological effects of metabolites Goal: to transform a lipophilic compound into a more hydrophylic species (metabolite) that can be excreted in urine or bile.

16. Metabolism of PCB 3 CYP450: Formation of arene-oxide CYP450: Formation of arene-oxide Sequential formation of mono- and dihydroxy metabolites Sequential formation of mono- and dihydroxy metabolites Preferred oxidation if two geminal H-atoms are present Preferred oxidation if two geminal H-atoms are present  Episodic congeners (e.g. PCB 3) versus “persistent” congeners (e.g. PCB 153) From: McLean et al., Chem. Res. Toxicol. 1996

17. Quinone Metabolites and Glutathione Adducts PCB quinones react with S- and N-nucleophiles, e.g. glutathione and glycine (Amaro et ai., Chem. Res. Toxicol. 1996) (Amaro et ai., Chem. Res. Toxicol. 1996) Catechol and hydroquinone PCB metabolites are oxidized to reactive quinones.

18. Reactive PCB Metabolites PCB ortho- and para-quinones: PCB ortho- and para-quinones: Arene oxide: Arene oxide: Semiquinone radical: Semiquinone radical:

19. PCBs and Carcinogenesis PCBs are complete carcinogens in rodents PCBs are complete carcinogens in rodents  Initiating (DNA damaging) activity has not been demonstrated Questions: Are reactive intermediates of PCB metabolism involved? Are reactive intermediates of PCB metabolism involved?or Are endogenous reactive intermediates generated by PCBs involved? Are endogenous reactive intermediates generated by PCBs involved?

20. Formation of DNA Adducts In Vitro Experimental approach to study DNA adduct formation in vitro: Experimental approach to study DNA adduct formation in vitro: Benzo- and hydroquinone adduct pattern are similar Benzo- and hydroquinone adduct pattern are similar Adduct pattern/reactivity dependent of Cl-substitution: 2-Cl >>> 3,4,5-triCl Adduct pattern/reactivity dependent of Cl-substitution: 2-Cl >>> 3,4,5-triCl Cytosine, adenine and thymidine adducts, but no guanine adducts (guanine is a reactive base!) Cytosine, adenine and thymidine adducts, but no guanine adducts (guanine is a reactive base!) Benzo- and hydroquinone PCBs covalently bind to DNA Benzo- and hydroquinone PCBs covalently bind to DNA Arif et al., Chem.-Biol. Int., 2003

21. PCBs as Initiators In Vivo Initial Studies used the Solt-Farber Protocol: Initial Studies used the Solt-Farber Protocol: PCB 3, 15, 52 and 77 increase number of GGT positive fociPCB 3, 15, 52 and 77 increase number of GGT positive foci PCB 3 and 15 increase volume of GGT positive fociPCB 3 and 15 increase volume of GGT positive foci  PCB 3 metabolites in a modified Solt-Farber protocol (fasting) Espandiari et al., TAP, 2003 GGT =  -glutamyltranspeptidase, PH = partial hepatectomy, AAF = acetylaminofluorene

22. PCB 3 Metabolites as Initiators Metabolites of PCB 3 studied: Metabolites of PCB 3 studied: –Monohydroxy –Dihydroxy –Ortho and para quinones GGT positive foci formed by 4-OH and 3,4-diOH PCB 3 GGT positive foci formed by 4-OH and 3,4-diOH PCB 3 3,4-Benzoquinone: ultimate carcinogen 3,4-Benzoquinone: ultimate carcinogen

23. Colvalent Binding of PCB Metabolites to Proteins Protein binding: Protein binding: –Inhibition of topoisomerase by PCB quinones in vitro Srinivasan et al., CRT, 2002 –Hemoglobin (in mice): Binding of PCB 4 is 10fold greater compared to PCB 77 Tampal et al., Toxicol. Lett., 2003 –Binding of PCB 3 and PCB 77 to proteins (in mice) Pereg et al., Chem.-Biol. Int., 2001

24. Endogenous Reactive Intermediates: ROS Dihydroxy-PCBs produce superoxide Dihydroxy-PCBs produce superoxide Quinones produce superoxide in the presence of GSH Quinones produce superoxide in the presence of GSH DNA strandbreaks caused by hydroxyl radicals and singlet oxygen DNA strandbreaks caused by hydroxyl radicals and singlet oxygen ROS production in cells in culture (HL-60) ROS production in cells in culture (HL-60) Putative generation of ROS by dihydroxylated PCB metabolites Srinivasan et al., Tox. Sci., 2001

25. Metabolism of Xenobiotics PHASE I METABOLISM Introduction or exposure to a functional group by oxidative or reductive processes or by hydrolysis (-OH, -NH 2, -SH, -COOH) e.g.: Cytochrome P450 (microsomes) PHASE II METABOLISM Reaction of a cofactor with an existing or created functional group e.g.: glucuronide conjugation Biological effects of metabolites Goal: to transform a lipophilic compound into a more hydrophylic species (metabolite) that can be excreted in urine or bile.

26. Hydroxylated PCBs in Humans: Blood, Adipose Tissue and Liver Blood: Blood: OH group in 3- or 4-position OH group in 3- or 4-position Cl-atoms vicinal to OH group Cl-atoms vicinal to OH group Concentrations of same magnitude as persistent PCB congeners Concentrations of same magnitude as persistent PCB congeners Most abundant metabolites: Most abundant metabolites: Adipose tissue and liver: Adipose tissue and liver:  OH-PCB Liver >  OH- PC adipose  OH-PCB Liver >  OH- PC adipose Correlation between  metabolites and  PCBs Correlation between  metabolites and  PCBs  metabolites vs.  PCBs:  metabolites vs.  PCBs: – Liver: 3 to 26% of  PCBs – Adipose: 0.3 to 0.8%  PCBs Predominant metabolites: Predominant metabolites: Berman et al., EHP, 1994Guvenius et al., Environ Toxicol Chem, 2002

27. Glucuronidation of OH-PCBs with Glucuronidation of OH-PCBs with uridine diphosphate glucuronosyl transferase (UGT) V max /K m : <3 to 116 L/min/mg Dependent on the structure of the metabolites: 2 nd ring: Cl-substituents lowered 2 nd ring: Cl-substituents lowered V max 2 nd ring: 2 nd ring: Substitution in 3- and 4- positions least favorable plays no role Steric hindrance around the hydroxyl group plays no role Enzymatic activity ~ surface area and surface volume Enzymatic activity ~ surface area and surface volume From: Tampal et al., Chem. Res. Toxicol. 2002 Efficiency of Glucuronidation (Vmax/Km): 2 1

28. Inhibition of Phase II Enzymes from Catfish by PCB Metabolites Inhibitors of sulfotransferase (dependent on position of OH- group) Inhibitors of sulfotransferase (dependent on position of OH- group) Inhibitors of glucuronosyltransferase Inhibitors of glucuronosyltransferase  Consequences for bioavailability, bioaccumulation and toxicity of other phenolic environmental contaminants 3-Hydroxy-benzo[a]pyrene BaP-3-OH) BaP-3-glucuronide BaP-3-sulfate Glucuronosyl transferase Phenol-type sulfotransferase OH-PCB Van den Hurk et al., EHP, 2002

29. Receptor-Enzyme Interactions for PCBs & Metabolites Aryl hydrocarbon (AhR)“coplanar” PCBs Constitutive androstane (CAR) ortho/para PCBs Estrogen (ER)OH-PCBs Ryanodine (RyR) PCB 95, OH-PCBs, catechols Phase II enzymes OH-PCBs Tyrosine hydroxylasemulti-ortho PCBs Binding sites PCBs & OH-PCBs on carrier proteins(Vit A, thyroid) Others??? Others???

30. Other… Nongenotoxic modes of action Nongenotoxic modes of action Effects on estradiol secretion by ovarian folicles Effects on estradiol secretion by ovarian folicles Neurotoxicity of PCB enantiomers Neurotoxicity of PCB enantiomers Effects of PCBs on thyroid hormone action Effects of PCBs on thyroid hormone action DNA damage by endogenous mechanism, e.g. ROS (reactive oxygen species) DNA damage by endogenous mechanism, e.g. ROS (reactive oxygen species)

31. Conclusions PCBs are activated to reactive intermediates that bind to cellular macromolecules (proteins, DNA) PCBs are activated to reactive intermediates that bind to cellular macromolecules (proteins, DNA)  Initiators of Carcinogenesis PCB metabolites interact with a large variety of receptors: PCB metabolites interact with a large variety of receptors:  PCB metabolites are the big unknowns in PCB toxicity

32. Acknowledgements Collaborators: Collaborators: –J. Arif –E. Gregoraszczuk –R.C. Gupta –M. James –G. Ludewig –M. Machala –L. W. Robertson –S. Parkin –… Students and postdocs: Students and postdocs: –A. Amaro –P. Espandiari –M. McLean –D. Pereg –N. Tampal –A. Srinivasan –…