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Prostaglandin H synthase catalyzes the oxidation of 4-chlorobiphenyl metabolites, and the in vivo effects on prostaglandin production

ProQuest Dissertations and Theses, 2011
Dissertation
Author: Orarat Wangpradit
Abstract:
Polychlorinated biphenyls (PCBs) exert a broad range of toxicity via both parent compounds and their metabolites. Our previous study showed that hydroquinone (H2 Q) metabolites of PCBs act as cosubstrates for prostaglandin H synthase (PGHS), and are oxidized by this enzyme to corresponding quinones (Q). The goal of this thesis is to illuminate the PGHS-mediated toxicity of lower chlorinated PCBs. It is hypothesized that PGHS catalyzes two sequential one-electron oxidations of PCB-H 2 Q to semiquinone (SQ*- ), and Q that interact with biomolecules, such as amino acids, glutathione (GSH), protein, and DNA. In addition, the oxidation of H2 Q by PGHS results in an elevation of downstream prostaglandin (PG) production in vivo. Employing 4-chlorobiphenyl-2',5'-hydroquinone (4-CB-2',5'-H2 Q) as a model compound, I found that PGHS-2 catalyzes the one-electron oxidation of 4-CB-2',5'-H2 Q to SQ*- . An unusual electronically distorted SQ*- spectrum was observed as a result of the mixture of two different SQ*- species, a quartet and a doublet. Fate of 4-CB-2',5'-SQ*- and/or Q in the presence of biomolecules was further investigated in the next study. 4-CB-2',5'-SQ *- /Q reacts readily with the thiol-containing molecules, such as cysteine, and GSH. Oligonucleotides, and DNA did not form a covalent adduct with 4-CB-2',5'-SQ*- but preferably stabilized 4-CB-2',5'-SQ *- by π-stacking interaction under the assay conditions. The in vivo study of downstream PG production in rats treated with 4-CB-2',5'- H2 Q revealed that PGE2 was significantly elevated in rats' kidneys at 24 h post intratracheal instillation. The increased PGE2 production was correlated with an elevation of alveolar macrophages. These findings suggest two possible mechanisms of enhanced PGE2 production: i) 4-CB-2',5'-H 2 Q as a cosubstrate for PGHS in kidney, and 2) release of cytokines from macrophages, leading to stimulation of PGE2 production in other tissues but released and accumulated in kidney for excretion. In summary, the toxicity of lower chlorinated PCBs metabolites is potentially mediated by PGHS. Quinones generated from the PGHS metabolic pathway covalently bind to GSH resulting in GSH depletion, and oxidative stress. The intercalation or π-stacking of SQ *- in DNA may be implicated in genotoxicity as a result of the change in DNA structure.

TABLE OF CONTENTS

CHAPTER 1 BACKGROUND

AND SIGNIFICANCE

.....................................................1

1.1 Polychlorinated biphenyls (PCBs)

..............................................................1 1.1.1 The origin, uses, and environmental fate of PCBs

...........................1 1.1.2 Toxicity of PCBs

..............................................................................3 1.1.3 Biotransforma tion of PCBs

..............................................................5 1.1.4 Mechanism of toxicity

......................................................................7 1.2 Prostaglandin H synthase (PGHS)

..............................................................9 1.2.1 Overall structures and properties

....................................................10 1.2.2 Biosynthesis of prostanoids and their functions

.............................11 1.2.3 Mechanism of PGHS catalysis

.......................................................13 1.2.4 Xenobiotic cooxidation ..................................................................16 1.3 Overview of thesis

....................................................................................18 CHAPTER 2 ONE - ELECTRON OXIDATION O F PCB METABOLITES TO

SEMIQUINONE RADICALS

BY PGHS - 2

..................................................21

2.1 Introduction

...............................................................................................21 2.2 Materials and methods

..............................................................................22 2.2.1 O xidation of 4- CB - 2’,5’ - H 2 Q by hPGHS - 2

...................................23 2.2.2 Formation of 4- CB - 2’,5’ - SQ ● -

in the presence of S - flurbiprofen, a cyclooxygenase inhibitor

.................................................23 2.2.3 Effect of slow tumbling and internal rotation energy

.....................24 2.2.4. Degradation of hPGHS - 2 side - chain by pronase

...........................24 2.2.5 In silico

molecular docking

............................................................24 2.2.6 Quantum mechanical calculations

..................................................26 2.3 Results and discussion ..............................................................................27 2.3.1 O xidation of 4- CB - 2’,5’ - H 2 Q by hPGHS - 2

...................................27 2.3.2 Formation of 4- CB - 2’,5’ - SQ ● -

in the presence of S - flurbiprofen , a cyclooxygenase inhibitor

.................................................29 2.3.3 Effect of slow tumbling and internal rotation energy

.....................30 2.3.4. Degr adation of hPGHS - 2 side - chain by pronase

...........................31 2.3.5 In silico

molecular docking

............................................................31 2.3.6 Quantum mechanical calculations

..................................................32 2.4 Conclusions ...............................................................................................33 CHAPTER 3 INTERACTIO N OF PCB - SEMIQUINONE WITH BIOMOLECULES

.........................................................................................47

3.1 Introduction

...............................................................................................47 3.2 Materials and methods

..............................................................................48 3.2.1 EPR determination of 4- CB - 2’,5’ - SQ ● -

in the presence of biomolecules

............................................................................................49 3.2.2 Oxygen consumption ......................................................................50 3.2.3 Quantification of GS - adducts by LC - MS

.......................................51 3.2.4 NMR determination of adduct formation .......................................51 3.2.5 Semi - empirical computations of the complexes between 4 - CB - 2’,5’ - SQ ● - and dinucleotides

.............................................................52 3.3 Results and discussion ..............................................................................53 3.3.1 DNA, oligonucleotides, and deoxynucleosides

..............................54

vi

3.3.2 Glutathione (GSH)

..........................................................................55 3.3.3 Amino acids

....................................................................................57 3.4 Conclusions ...............................................................................................58 CHAPTER 4 CHARACTERI ZATION OF THE QUINON E ADDUCTS

.......................70

4.1 Introduction ...............................................................................................70 4.2 Materials and methods

..............................................................................71 4.2.1 Identification of quinone - NAC adducts

.........................................71 4.2.2 Characterization of the adducts by NMR

.......................................72 4.2.3 Identification of the quinone - BSA adduct

......................................73 4.3 Results and discussion ..............................................................................74 4.3.1 Identification and characterization of the quinone - NAC adducts

.....................................................................................................74 4.3.2 Identification of the quinone - BSA adduct

......................................75 4.4 Conclusions ...............................................................................................76 CHAPTER 5 PCB METABO LITES ALTER THE PROD UCTION OF PROSTAGLANDIN IN VIVO

........................................................................84

5.1 Introduction

...............................................................................................84 5.2 Materials and methods

..............................................................................86 5.2.1 Chemicals

and experimental devices

..............................................86 5.2.2 Uptake of 4- CB metabolites by PAMAM dendrimers

...................86 5.2.3 Animals

...........................................................................................87 5.2.4 Intratracheal instillation (ITI)

.........................................................87 5.2.5 Evaluation of bronchoalveolar lavage (BAL) fluid ........................87 5.2.6 Extraction of PGE 2

metabolites from rats’ organs

.........................88 5.2.7 Determination of PGE 2

metabolites by EIA

..................................88 5.2.8 Statistical analysis

..........................................................................89 5.3 Results and discussion ..............................................................................90 5.3.1 Uptake of 4- CB metabolites by PAMAM dendrimers

...................90 5.3.2 Bronchoalveolar lavage (BAL) fluid ..............................................91 5.3.3 PGE 2 production in rats’ organs

.....................................................92 5.4 Conclusions ...............................................................................................94 CHAPTER 6 SUMMARY AND FUTURE PERSPECTIVE S

.......................................100

6.1 Prostaglandin H synthase - mediated PCB toxicity

..................................100 6.2 Implication of PCBs in inflammatory response

......................................103 6.3 Future perspectives

.................................................................................104

REFERENCES

................................................................................................................106

vii

LIST OF TABLES

Table 2 - 1: The analysis of the nuclei - centered electron density [q(A)] obtained from the calculation of the quantum theory of atoms in molecules (QTAIM) in the presence (prop), or absence (nonprop) of the propionate molecule.

..........................................................................................................42 Table 2 - 2: Diatomic electron pair contribu tion to bonding (an extract of the most relav a nt information obtained from the QTAIM calculations in the absence (noprop) and presence (prop) of the propionate molecule.

................44 Table 4 - 1 : 1 H and 13 C NMR results of 4 - CB - 2’,5’ - H 2 Q - 4’ - NAC and 4 - CB - 3’,4’ - H 2 Q - 6’ - NA C adducts listed as chemical shifts (δ) determined in ppm relative to TMS and coupling constants (J).. ...................................................83

viii

LIST OF FIGURES

Figure 1 - 1: Chemical structure and numbering of carbon atoms for PCBs.

........................1

Figure 1 - 2: Metabolic pathway of 3,4- dichlorobiphenyls (3,4 - DCB)..

...............................6

Figure 1 - 3: Formation of SQ ● -

and ROS from the redox cycling of H 2 Q and Q.

................9

Figure 1 - 4: Biosynthesis of prostanoids . ...........................................................................12

Figure 1 - 5: Mechanism of PGHS catalysis.

.......................................................................15

Figure 1 - 6: Three dimensional structure of ovine PGHS - 1 (oPGHS - 1).

...........................16

Figure 2- 1: Numbering of 4 - CB - 2’,5’ - H 2 Q and heme - propionate subsystem used in the quantum mechanics studies. .....................................................................27

Figure 2 - 2: Proposed mechanism of oxidation of 4- CB - 2’,5’ - H 2 Q and its fluoro - substituted analogues (1) , to mesomeric 4 - CB - 2’,5’ - SQ ● -

(2a - c) by PGHS.

.............................................................................................................34

Figure 2 - 3: EPR spectra of 4 - CB - 2’,5’ - SQ ● - formation. ...................................................35

Figure 2 - 4: Simulation of an EPR spectrum from the incubation of 4- CB - 2’,5’ - H 2 Q with hPGHS - 2, hematin and KAA (complete incubation). ............................36

Figure 2 - 5: EPR spectra of 4 - CB - 2’,5’ - SQ ● - formed in the incubation of 4- CB - 2’,5’ - H 2 Q with different concentrations of hPGHS - 2. ..................................37

Figure 2 - 6: EPR spectra of 4 - CB - 2’,5’ - SQ ● - in the incubation of 4- CB - 2’,5’ - H 2 Q with hPGHS - 2, in the presence of S - flurbiprofen, a cyclooxygenase inhibitor.

.........................................................................................................38

Figure 2 - 7: EPR spectra

of 4 - CB - 2’,5’ - SQ ● -

generated from the reaction mixture of 4- CB - 2’,5’ - Q in 50% glycerol (blue), and 2- F - 4 - CB - 2’,5’ - SQ ● -

generated from 2 - F - 4 - CB’2’,5’ - Q in PBS (red).

............................................39

Figure 2 - 8: EPR spectra of 4 - CB - 2’,5’ - SQ ● -

from the incubation of 4- CB - 2’,5’ - H 2 Q with p ronase - treated hPGHS - 2.. ............................................................40

Figure 2 - 9: Most favorable binding mode for 4- CB - 2’,5’ - H 2 Q within the peroxidase site of hPGHS - 2.. .........................................................................41

Figure 3 - 1: EPR spectra of 4- CB - 2’,5’ - SQ ● - formed in the incubation of 100 µM 4- CB - 2’,5’ - H 2 Q with 200 units hPGHS - 2 in the presence of salmon sperm DNA (SSDNA).

...................................................................................60

Figure 3 - 2: Oxygen consumption of the incubation of 50 µM 4- CB - 2’,5’ - H 2 Q in the presence of SSDNA and GSH.

.................................................................61

ix

Figure 3 - 3: EPR spectra of 4 - CB - 2’,5’ - SQ ● -

formed in the incubation of 100 µM 4- CB - 2’,5’ - H 2 Q with 200 uni ts hPGHS - 2 in the presence of 50 µM oligonucleotides (20 bases). ...........................................................................62

Figure 3 - 4: EPR spectra of 4 - CB - 2’,5’ - SQ ● -

formed in the incubation of 100 µM 4- CB - 2’,5’ - H 2 Q with 200 units hPGHS - 2 in the presence of 100 µM deoxynucleosides, dA, dC, dG, and dT. .........................................................63

Figur e 3 - 5: The optimized structures of the complexs of dinucleotides (dApdA, dGpdG and dTpdT) and 4- CB - 2’,5’ - SQ ● -

(left), and the mesh images of singly occupied molecular orbitals (SOMOs) of the complexes (right).

.......64

Figure 3 - 6: EPR spectra of 4 - CB - 2’,5’ - SQ ● -

formed in the incubation of 100 µM 4- CB - 2’,5’ - H 2 Q

with 200 units hPGHS - 2 in the presence of GSH. .................65

Figure 3 - 7:

1 H NMR spectra of the adducts from the incubations of 4- CB - 2’,5’ - SQ ● -

/Q with dG, or GSH. The 4- CB - 2’,5’ - SQ ● -

was generated from comproportionat ion of an equal molar of 4- CB - 2’,5’ - H 2 Q and 4- CB - 2’,5’ - Q.

...........................................................................................................66

Figure 3 - 8: Oxygen consumption of the incubations of 50 µM 4- CB - 2’,5’ - H 2 Q

in the presence of GSH (no hPGHS - 2 added).

...................................................67

Figure 3 - 9: EPR spectra of 4 - CB - 2’,5’ - SQ ● -

formed in the incubations of 100 µM 4- CB - 2’,5’ - H 2 Q with 200 units hPGHS - 2 in the presence of 100 µM amino acids.

....................................................................................................68

Figure 3 - 10: Oxygen consumption of the incubation of 50 µM 4- CB - 2’,5’ - H 2 Q in the presence and absence of amino acids.

......................................................69

Figure 4 - 1:

1 H aromatic and peptide (NAC - α

and NAC - β ) spectral regions of different fractions (F) of 4 - CB - 2’,5’ - H 2 Q - NAC and 4 - CB - 3’,4’ - H 2 Q - NAC.

...............................................................................................................78

Figure 4 - 2:

1 H aromatic and peptide spectral regions (upper) and HMBC cross - section of 4- CB - 3′,4′ - H 2 Q - 5′ - NAC (lower).

..................................................79

Figure 4 - 3: Changes of the 13 C

NMR shifts, ∆ δ (ppm) in the 4- CB - 2’,3’ - Q, 4 - CB - 3’,4’ - Q, and 4- CB - 2’,5’ - Q expressed in relative values to 4 - CB.

..................80

Figure 4 - 4: Chromatograms of the incubation of 4 - CB - 2’,5’ - Q with BSA (upper) compared with the control (lower) showing no adduct formation.

................81

Figure 4 - 5: EPR spectra of 4 - CB - 2’,5’ - SQ ● - formed in the incubation of 4- CB - 2’,5’ - Q with different concentrations of bovine serum albumin (BSA).

.......82

Figure 5 - 1: Structure of a PAMAM dendrimer.

................................................................96

Figure 5 - 2: UV absorbance values of 4 - CB - 2’,5’ - H 2 Q (A), and 4- CB - 2’,5’ - Q (B) in the presence of dendrimers, compared to the control (DMSO). ....................97

Figure 5 - 3: Cell differentiations from bronchoalveolar lavage (BAL) fluid of rats necropsied at 4 and 24 h post exposure. .........................................................98

x

Figure 5 - 4: Concentration of PGE 2

metabolites f rom organs of rats necropsied at 4 and 24 h post exposure. ..................................................................................99

1 CHAPTER 1

BACKGROUND AND SIGNI FICANCE

1.1

Polychlorinated biphenyls (PCBs)

PCB s

are a group of synthetic organic compounds with 209 congeners

distinguished by the number and positions of chlorine atoms on the biphenyl rings . For example , 4- chlorobiphenyl (4 - CB) is a congener con taining

one chlorine substituent at the "4" carbon (para) position of the biphenyl ring. The chemical structure and numbering of carbon atoms for PCB s

is shown in Figure 1 - 1. Appearance of individual PCB congeners can vary from colorless crystals to viscous liquids at room temperature depending on the degree of chlorination ( UNEP, 1999 ) . Cl n H (10-n) 2 2' 3 3' 4 4' 5 5' 6 6'

Figure 1 - 1 : Chemical structure and numbering of carbon atoms for PCBs.

1.1.1 The origin, uses, and environmental fate of PCBs

PCB s

were

commercially produced in the United States from 19 29

until the late 1970s , and were marketed as complex mixtures. Aroclor ®

products were well - known PCB mixtures manufactured by Monsanto, USA, that were accounted for about 60% of the PCB production worldwide ( WHO, 1993 ) . Commercial PCB mixtures were widely used in a variety of applications, including transformers, capacitors, flame retardants, inks, adhesives, plasticizers, sealant s, oil - based paints, and cable insulation ( ATSDR, 2000 ; Erickson & Kaley, 2011 ; Fiedler, 2001 ) . The uses of PCB s were largely based on

2 their chemical stability and low flammability. Many Aroclor ®

PCB commercial mixtures are viscous liquids, although the majority of the individual PCB present are solids at room temperature. The greater the chlorine content, the grea ter their resistance to chemical and biological break - down ( Erickson, 1986 ) . Due to the concerns over toxicity and chemical stability of PCBs, which leads to their environmental persistence, the United States Enviromental Protection Agency (EPA) banned the domestic production of PCBs in 1979 ( EPA, 1979 ) . However, the use of PCBs in ‘completely closed’ applications such as capacitors and tranformers is still allowed ( EPA, 2010 ) . Releases o f PCBs into the environment nowsdays may result from the improperly maintained waste sites that contain PCBs, leaks from electrical transformers, and disposal of PCB - containing products ( EPA, 2010 ; Kimbrough & Krouskas, 2003 ) .

Although PCB s

are no longer produced and used in the manufactu re of new products, they are formed as by - products in dyes used in printing and paints ( Hu & Hornbuckle, 2010 ) . PCBs from historic use and disposal remain in soil, water and air for very long periods of time, due to their resistance to chemical and biological breakdown . PCBs can be deposited to t he areas far away from where they were released ( EPA, 2010 ) . Generally, the higher chlorinated PCB congeners tend to persist in the environment longer than the lower chlorinated PCBs which are more capable of movement due to their higher vapor pressures ( Lang, 1992 ; Safe, 1994 ) . Long distance movement of PCBs was first realized in 1966 when Jensen discovered PCBs in the Arctic ( Jensen, 1966 ) . The persistence of PCBs in the ecosystem leads to an accumu lation of these chemicals in the food chain, especially in fish and marine mammals due to their lipophilicity and tendency to bio- accumulate ( ATSDR, 2000 ) .

3 1.1.2 Toxicity of PCBs

Exposure to PCBs occur s

via

dermal contact, consuming PCB - contaminated food, and inhaling contaminated air.

A number of human health effects resulted from PCB expos ure include dermatologic, reproductive, developmental, hepatic, immunological, and carcinogenic effects ( ATSDR, 2000 ) . Dermatologic effects : Two well - known incidents, Yusho in Japan (1968) and Yu - Cheng in Taiwan (1979) caused by accidental consumption of rice oils that were contaminated by heat - degraded PCBs during processing, underscored th e toxicity of PCBs to human health. The toxic effects seen in these populations included chloracne, ocular lesions, hyperpigmentation of skin, conjunctivae, and nails ( Aoki, 2001 ) . Chloracne is the only overt sign of PCB exposure in humans that indicates both local effects from dermal contact and systemic effects from ingestion of PCBs ( Gehle et al. , 2000 ) . Reproductive effects:

A recent study by Bonde et al.

( Bonde et al. , 2008 )

showed that low sperm count and decreased sperm motility were observed in male humans showing increased PCB - 153 concentrations in their serum. Female reproductive system is also in terfered with PCB exposure. Irregular menstrual cycle was found in female populations of Yusho and Yu - Cheng incidents ( Aoki, 2001 ) . Similar evidence was noted with reduced menstrual cycle length in Swedish women exposed to high levels of PCBs ( Cooper et al. , 2005 ) .

Developmental effects:

Exposure to PCBs during the prenatal and lactation peroids can cause developmental deficiencies in newborns ( Arena et al. , 2003 ; Ribas - Fito et al. , 2001 ) . Developmental effects in the children of mothers who consumed moderate to high amounts of PCB - contamina ted Lake Michigan fish during pregnancy included decreases in gestational age, birth weight, and head circumference ( Fein et al. , 1984 ) . A follow - up study revealed that these children at 11 years of age were three times more likely than controls to have low full - scale verbal IQ scores, and difficulty paying

4 attention ( Jacobson & Jacobson, 1996 ) . Similar neurodevelopmental deficits in children born to the pregnant women during the Yu - Cheng incident also persisted for several years after initiation of PCB exposure ( Chen et al. , 1992 ; Rogan et al. , 1988 ) . Hepatic effects:

The primary change in the liver of PCB - exposed populations is an induction of liver enzymes, especially cytochrome P450 (CYP) enzymes, which is thought to be a sensitive marker for hepatic change observed in PCB - exposed animals ( ATSDR, 2000 ) . The mechanism of CYP induction by PCBs will be explained in section 1.1.4 . Although no evidence of overt hepatotoxicity has been found in humans, the incidence of cirrhosis and asymptomatic hepatomegaly was reported in workers who were exposed and had concomitantly high PCB levels in serum ( Hsu et al. , 1985 ) . Yusho victims have shown transient increases in hepatocellular carcinoma ( Okumura, 1996 ) .

Immunological effects:

Immunosuppression is a general effect found in PCB - exposed populations. The immosuppressive effects include decreases in natural killer cells, antibody levels, monocyte

and granulocyte counts in humans ( Wei sglas - Kuperus et al. , 1995 ) , and decreases in size of thymus gland in Rhesus monkeys ( EPA, 2008 ) . It has also been noted that Yusho and Yu - Cheng populations and the newborns of mothers with high serum PCB levels were more susceptible to respiratory infections ( Gehle et al. , 2000 ; Nakanishi et al. , 1985 ) . Carcinogenic effects:

PCBs were classified as a probable human carcinogens, group 2A, by the International Agency for Research on Cancer (IARC) due to the fact that PCBs cause hepatocarcinomas, lymphomas, leukemia, and tumors in animal models ( ATSDR, 2000 ; Gehle et al. , 2000 ; IARC, 1987 ) . A few studies in workers reported that occupational exposure to PCBs increased risk of cancer in liver, gall bladder, and biliary tract ( Brown & Jones, 1981 ; Gustavsson et al. , 1986 ) . More recent cohort studies showed that increased PCB concentrations in blood were associated with increased risk of non - Hodgkin lymphoma ( Engel et al. , 2007 ; Rothman et al. , 1997 ) . Carcinogenesis is a multistage process composed of initiation, promotion and progression. Various higher

5 chlorinated PCBs have been reported to possess promoting activitiy in rat liver model ( Glauert et al. , 2008 ; Silberhorn et al. , 1990 ) , whereas lower chlorinated PCBs need enzymatic biotransformation to hydroxylated metabolites and act as initiators in the carcinogenesis

( Espandiari et al. , 2004 ; Srinivasan et al. , 2001 ; Xie et al. , 2010 ) . The biotransformation of PCBs and mechanism of toxicity will be further explained in sections 1.1.3 and 1.1.4 , respectively.

1.1.3 Biotransformation of PCBs

The toxicity of certain PCBs may be found in the formation of their reactive metabolites. Therefore, the biotransformation of PCBs, particularly lower chlorinated PCBs may be impor tant to consider regarding

their toxicity. PCBs are generally stable, and some congeners ( i.e. ,

ortho - substituted higher chlorinated PCBs) are resistant to metabolism ( James, 2001 ) . However, the general metabolic pathways of PCBs are quite similar for all congeners as shown in Figure 1 - 2 . 3,4 - Dichlorobiphenyl is used in this figure as a representative for PCBs that can be metabolically

activated.

The first step in PCB metabolism is the introduction of an oxygen atom by CYP into the biphenyl ring. Particular CYP isozymes can catalyze the mono - oxygenation of certain PCB congeners depending on their chlorine substitution patterns. The cata lysis rate of mono - oxygenation of PCBs by CYPs is inversely related to the degree of chlorination. The higher chlorinated PCBs are more resistant to CYP - mediated metabolism than the lower chlorinated ones ( Sundstrom, 1976 ) . The metabolism of PCBs by CYPs results in m onohydroxy metabolites as the major products from the isomerization of an arene oxide intermediate or the direct insertion of a hydroxy group to the biphenyl ring. Lower chlorinated biphenyls may directly undergo two successive hydroxylation steps to yield

Full document contains 140 pages
Abstract: Polychlorinated biphenyls (PCBs) exert a broad range of toxicity via both parent compounds and their metabolites. Our previous study showed that hydroquinone (H2 Q) metabolites of PCBs act as cosubstrates for prostaglandin H synthase (PGHS), and are oxidized by this enzyme to corresponding quinones (Q). The goal of this thesis is to illuminate the PGHS-mediated toxicity of lower chlorinated PCBs. It is hypothesized that PGHS catalyzes two sequential one-electron oxidations of PCB-H 2 Q to semiquinone (SQ*- ), and Q that interact with biomolecules, such as amino acids, glutathione (GSH), protein, and DNA. In addition, the oxidation of H2 Q by PGHS results in an elevation of downstream prostaglandin (PG) production in vivo. Employing 4-chlorobiphenyl-2',5'-hydroquinone (4-CB-2',5'-H2 Q) as a model compound, I found that PGHS-2 catalyzes the one-electron oxidation of 4-CB-2',5'-H2 Q to SQ*- . An unusual electronically distorted SQ*- spectrum was observed as a result of the mixture of two different SQ*- species, a quartet and a doublet. Fate of 4-CB-2',5'-SQ*- and/or Q in the presence of biomolecules was further investigated in the next study. 4-CB-2',5'-SQ *- /Q reacts readily with the thiol-containing molecules, such as cysteine, and GSH. Oligonucleotides, and DNA did not form a covalent adduct with 4-CB-2',5'-SQ*- but preferably stabilized 4-CB-2',5'-SQ *- by π-stacking interaction under the assay conditions. The in vivo study of downstream PG production in rats treated with 4-CB-2',5'- H2 Q revealed that PGE2 was significantly elevated in rats' kidneys at 24 h post intratracheal instillation. The increased PGE2 production was correlated with an elevation of alveolar macrophages. These findings suggest two possible mechanisms of enhanced PGE2 production: i) 4-CB-2',5'-H 2 Q as a cosubstrate for PGHS in kidney, and 2) release of cytokines from macrophages, leading to stimulation of PGE2 production in other tissues but released and accumulated in kidney for excretion. In summary, the toxicity of lower chlorinated PCBs metabolites is potentially mediated by PGHS. Quinones generated from the PGHS metabolic pathway covalently bind to GSH resulting in GSH depletion, and oxidative stress. The intercalation or π-stacking of SQ *- in DNA may be implicated in genotoxicity as a result of the change in DNA structure.