GENDER DIFFERENCES IN ALPHA-TOCOPHEROL PROTECTION AGAINST BENZO[ A ]PYRENE-INDUCED DAMAGE IN RATS

GENDER DIFFERENCES IN ALPHA-TOCOPHEROL PROTECTION AGAINST BENZO[A]PYRENE-INDUCED DAMAGE IN RATS Caitlin Crimp Mentor: Debbie Mustacich, Ph.D.

POLYCYCLIC AROMATIC HYDROCARBONS • PAHs are a class of environmental toxins produced by incomplete combustion processes. • High occupational exposures occur; i.e. road paving, roofing, second-hand smoke (bars, casinos), forest fires, smoke-houses (Boffetta ‘97). • High occupational PAH exposure entails a substantial risk of lung, skin, and bladder cancer (Boffetta ‘97).

BENZO[A]PYRENE • Benzo[a]pyrene (BP) is a common PAH found in most PAH mixtures. • BP was determined to be the component of coal tar responsible for scrotal cancer in chimney sweeps. Benzo[a]pyrene

ALPHA-TOCOPHEROL (α-T) • Of the eight forms of vitamin E, α-T is maintained at the highest concentrations in the tissues and plasma of humans due to the specificity of the α-tocopherol transfer protein (Traber ‘05). 2R 4’R 8’R RRR-alpha-tocopherol

ANTIOXIDANT ROLE • α-T is a lipid-soluble antioxidant that protects cell membranes by stopping the lipid peroxidation chain reaction. pentane α-TOH Ascorbate α-TO malondialdehyde Ascorbate ethane

BENZO(A)PYRENE & α-TOCOPHEROL • α-T supplementation decreases BP-DNA adduct levels in smokers. • Effects were greater in women than men (Mooney ‘05). • Effects were greatest in women with the GSTM1-null phenotype (Mooney ’05).

BENZO[A]PYRENE METABOLISM Adapted from Baird ‘05

STUDY GOAL The long-range goal of these studies is to determine the efficacy of prophylactic high dose vitamin E supplementation for prevention of DNA-adduct formation during occupational exposure to PAHs.

HYPOTHESIS Our working hypothesis is that elevated levels of α-T decrease BP-induced damage by two synergistic mechanisms: (1) increased antioxidant protection against oxidative stress-induced damage (2) modulation of BP metabolism and/or excretion. In addition, we hypothesize that this protection will be greater in female rats compared to males.

EXPECTED OUTCOMES α-T supplementation would: • Prevent BP-induced changes in α-T and reduced glutathione (GSH) levels • Prevent BP-induced increases of F2-isoprostanes, malondialdehyde (MDA), oxidized glutathione (GSSG), and oxo8dG • Alter the total radioactivity in tissues and/or plasma or urine • Offer greater protection in BP-treated female rats compared to BP-treated males.

EXPERIMENTAL DESIGN • Male and female Sprague Dawley rats (225-350g) were given 7 daily s.c. injections of either α-T or vehicle (10 mg α-T/100 g body wt). • On day 8 rats were i.p. injected with radiolabeled BP (20 mg BP/ kg body wt). • Urine was collected 24 h prior and 24 h following BP injection. • 24 h post BP injection, following a 12 h fast, rats were euthanized and tissues and plasma collected and stored at -80˚C until analysis.

METHODS • Plasma and tissue α-T were extracted as described by Podda ’96 and measured using HPLC-fluorescence detection • Total radioactivity was measured by LSC • Urine F2-isoprostanes: • extracted using the method described by Taylor ‘06 • measured by enzyme immunoassay (Cayman Chemical, measures 8-isoPGF2α) • normalized to creatinine, measured by the Jaffe reaction (Assay designs)

α-TOCOPHEROL SUPPLEMENTATION INCREASES α-TOCOPHEROL TISSUE AND PLASMA LEVELS IN BP TREATED RATS a = p < 0.05 between treatment groups b = p < 0.05 between sexes

α-TOCOPHEROL SUPPLEMENTATION ALTERS TOTAL RADIOACTIVITY IN THE TISSUES OF 3H-BP TREATED RATS 24 H POST TREATMENT a = p < 0.05 between treatment groups b = p < 0.05 between sexes

α-TOCOPHEROL SUPPLEMENTATION ALTERS TOTAL RADIOACTIVITY IN THE PLASMA AND URINE OF 3H-BP TREATED RATS 24 H POST TREATMENT a = p < 0.05 between treatment groups b = p < 0.05 between sexes

α-T SUPPLEMENTATION PREVENTS BP-INDUCED ELEVATION OF URINE 8-ISOPGF2α LEVELS 24 H FOLLOWING BP EXPOSURE IN RATS Pre-BP level a = p < 0.05 between treatment groups b = p < 0.05 between sexes

SUMMARY • α-T supplementation in BP-treated rats: • Increases tissue and plasma α-T levels • Effect was greater in females than males • Alters tissue, urine and plasma total radioactivity • Decreases in males with supplementation • Increases in Females with supplementation • Prevents BP-induced elevation of urinary 8-IsoPGF2α • Both males and females

CONCLUSIONS • BP does not deplete α-T tissue or plasma levels 24 h post treatment in supplemented or non-supplemented rats • α-T supplementation: • alters BP and/or BP metabolite levels in tissues, urine and plasma as indicated by total radioactivity • Prevents BP-induced lipid peroxidation as indicated by decreased urinary 8-isoPGF2α as compared to non-supplemented rats • α-T supplementation alters BP treatment outcomes in a gender-dependent manner in rats

FUTURE STUDIES • Complete tissue GSH and MDA analysis • Determine BP-DNA adduct and oxo8dG levels in tissues • Determine BP metabolites in tissues and urine • Clinical trials: α-T supplementation and occupational high level PAH exposure • In utero exposure studies in sheep

ACKNOWLEDGEMENTS • Funding: • NIEHS Grant 5R21ES015872 • Howard Hughes Medical Institute • URISC • Cripps Scholarship Fund • Debbie Mustacich, Ph.D. • Maret Traber, Ph.D. • Tammie McQuistan • Scott Leonard, M.S. • Alan Taylor, M.S. • Kevin Ahern, Ph.D. • Traber Lab • Baird Lab • Rats

GENDER DIFFERENCES IN ALPHA-TOCOPHEROL PROTECTION AGAINST BENZO[ A ]PYRENE-INDUCED DAMAGE IN RATS