Oxidative stress, the Lens Bends HealthCheryl Burdette, ND

Estrogen Metabolite Ratios: Time for Us to Let Goby Jacob Schor, ND, FABNO

Hormone Metabolism

• Do No Harm-a healthy system is the backdrop for safe hormone use

• Menopausal Symptom Relief• Get Side Benefits

• Coagulation• Bone Health• Anti-inflammatory• Anti-Oxidant• Cognitive• Cardiovascular

The Need

Take a History; Do an Exam; Test as needed; Look for Cause

Treat the Matrix:◦ Brain◦ GUT ◦ Detoxification Capacity◦ Mitochondrial Function/Oxidative Stress◦ Immune Balance◦ Structural Imbalance◦ Mind-Body-Spirit◦ Hormone Imbalance

THEN Treat Symptoms - OR Treat Symptoms WHILE searching for and ameliorating causes.

Copyright 2013

CONTEXT: How to Treat Hormone Imbalance

Relief for hot flushes, a predominant symptom of menopause is among the most common reasons for clinical visits of mid-life women and a major cost in health care expenditures. The median duration of moderate to severe hot flushes was 10.2 years…The most common ages at onset of moderate-to-severe hot flushes were 45–49 years

Hot flushes are associated with poor sleep, depressed mood, decreased quality of life, may worsen depressive symptoms and signal the onset or relapse of a major depressive episode.

Hot flushes may possibly mark underlying vascular changes that are associated with subclinical cardiovascular disease, increased aortic calcification among users of hormone therapy, greater incident coronary heart disease, and may be a risk factor for poor bone health.

“Hot flushes may possibly mark underlying vascular changes that are associated with subclinical cardiovascular disease, increased aortic calcification among users of hormone therapy, greater incident coronary heart disease, and may be a risk factor for poor bone health.”

Breaking the cycle of Dis-Ease means addressing the “Associated Risk Factors” as well. Recall from this research:

0%

60% 14% 79% 39%

6.5% in Premenopause, 56.6%

in Perimenopause, 50.7% in

Postmenopause

Prevalence of Hot Flashes

How common is the condition?Prevalence of

Insomnia

*Arch Intern Med. 2006;166:1262-1268Copyright 2013

Oxidative Stress Positive Correlation with Chronic Disease and Aging

Figure from Evans, JL et al (2000) Diabetes Technol Therap 2:401-413.

http://www.sodahead.com/fun/air-water-fire-or-earth-which-one-would-you-choose/question-3498247/?postId=103060425&page=2

Tier 1 Tier 2Tier 3

High GSH/GSSG ratio

Low GSH/GSSG ratio

Degrees of Oxidative Stress

Normal

Anti-oxidant Defens

e

Inflammation

Cytotoxicity

Response Pathway

Signaling Pathway

Nrf2 MAP kinase/Nf-κB cascade

Mitochondrial Permeability

Genetic Response

Anti-Oxidant Respons

e Element

Nf-kB

OutcomePhase II enzymes

Cytokines/chemokines Apoptosis

N/A

General◦ GSH/GSSG ratio; % GSH

Lipid◦ F2-isoprostanes◦ Lipid hydroperoxides ◦ Malondialdehyde (MDA), Thiobarbituric Acid (TBARS)

Protein◦ Protein carbonyls◦ Oxidized LDL (OxLDL)◦ Advanced glycation end-products (AGEs)◦ NF-kB (activated) ; inflammation

DNA◦ 8-hydroxy-2'-deoxyguanosine No consensus of DEFINITION of oxidative stress other than a statistically significant increase in one of these (or some other) markers compared to control

Biochemical Markers of Oxidative Stress

(Selected Examples)

2/20/2013

Antioxidant Response Element

J Obstet Gynaecol Res. 2012 Sep;38(9):1177-81. doi: 10.1111/j.1447-0756.2011.01842.x. Epub 2012 Apr 30.Oxidative stress measured by carbonyl groups level in postmenopausal women after oral and transdermal hormone therapy.Polac I, Borowiecka M, Wilamowska A, Nowak P.SourceDepartment of Gynaecology and Menopausal Disorders, Polish Mother's Memorial Hospital - Research Institute, Lodz, Poland. irekpolac@interia.plMenopause is associated with an increased risk of cardiovascular disorders, which are accompanied by oxidative stress. Our study was undertaken to determine whether oxidative stress in menopausal women could be reduced after six months of oral or transdermal hormonal therapy.MATERIAL AND METHODS: Carbonyl groups of proteins in blood plasma were estimated by sensitive ELISA method with anti-DNP antibodies. In this method, protein samples diluted in phosphate-buffered saline were adsorbed to wells of an ELISA plate and then reacted with dinitrophenylhydrazine (DNPH).RESULTS: Plasma protein carbonyl levels of postmenopausal women treated with o-HT and t-HT for six months (o-HT: 1.785 ± 0.31 nmol/mg; t-HT: 1.838 ± 0.33 nmol/mg) were lower when compared with the control group (2.232 ± 0.28 nmol/mg). There was no statistically significant difference in carbonyl levels between women after oral and transdermal HT (P = 0.149).CONCLUSION: Hormonal therapy reduces the level of carbonyl protein, a marker of oxidative stress, suggesting potential protective effect.

Hormone therapy and oxidative stress

Climacteric. 2013 Feb;16(1):104-10. doi: 10.3109/13697137.2012.660711. Epub 2012 Apr 24.HRT decreases DNA and lipid oxidation in postmenopausal women.Escalante Gómez C, Quesada Mora S.SourceDepartment of Gynecology, Hospital San Juan de Dios, San José, Costa Rica.AbstractBACKGROUND: Postmenopausal women have increased oxidative stress and decreased antioxidant status. Estrogen has great antioxidant capacity both in vitro and in vivo. Few authors have studied the effect that hormone replacement therapy (HRT) has on the oxidant and antioxidant status and none have studied the effect on DNA oxidation as a possible explanation for the aging process itself.AIM: The aim of this study was to evaluate both oxidation and antioxidation markers in postmenopausal woman and to determine the effects that HRT has on them.METHOD: Sixty-two postmenopausal women with similar biophysical characteristics were divided into three groups: (1) 18 not taking any HRT, (2) 20 receiving estrogen-only replacement therapy (ERT, conjugated equine estrogen), and (3) 22 receiving combined estrogen/progestin HRT (conjugated equine estrogen + medroxyprogesterone acetate). Specific molecular oxidative damage was detected by measuring 8- hydroxy-2-

deoxy guanosine (8-OH-2dG) (DNA damage), standardized thiobarbituric acid reactive substances (TBARS) (lipid damage) and protein carbonyl (proteins). Antioxidant enzyme activity was detected by measuring catalase activity, and total antioxidant status was measured using 1,1,difenil-2-picril hydrazil. Both ELISA and photometric methods were used.RESULTS: 8-OH-2dG levels were significantly lower in women who received combined HRT compared to women who did not receive HRT (ANOVA, p < 0.05). Lipid oxidation was significantly lower in women on ERT compared to women taking no HRT (ANOVA, p < 0.05). Pearson correlation showed that lipid oxidation decreased as the estradiol concentration increased within the study range (r = -0.362, p < 0.05). No statistical difference was noted for protein oxidation and catalase activity among the groups. No statistical difference was found for total antioxidant status between the groups (ANOVA).CONCLUSIONS:

HRT decreases oxidative damage to both DNA and lipids in postmenopausal women. Lipid oxidation status may be inversely related to estrogen levels in postmenopausal women.

Hormone therapy may reduce oxidative damage

Menopause can be defined as a condition of oxidative stress. Biomarkers of oxidative stress are higher post menopause

Studies also show that there is more oxidative stress in women who have more symptoms of menopause.

Glutathione drops off in menopause which may be part of the reason for aging, nor merely decline in hormones. The implications clinically are important because this would mean we could treat menopause with glutathione therapies rather than hormones. This would be a safer and more benign method of treatment.

Estrogens, even those that are bioidentical and taken exogenously are safer when in a high glutathione environment. That is to say, women on HRT should also consider glutathione therapies to reduce risk of HRT.

This is a missed piece of hormone therapy. Glutathione therapies such as DIM and sulforaphane therapies should be

considered as an adjunct for all HRT. It not only shifts a 2:16 ratio (of debatable importance), but increases glutathione which decreases 4-OH and favors production of 2-OH. Glutathione therapies should be considered alongside HRT.

Rational for Glutathione therapies in Post menopausal interventions

Menopause. 2012 Mar;19(3):361-7.

Menopause as risk factor for oxidative stress.

Sánchez-Rodríguez MA, Zacarías-Flores M, Arronte-Rosales A, Correa-Muñoz E, Mendoza-Núñez VM.

Source

From the 1Unidad de Investigación en Gerontología, Facultad de Estudios Superiores Zaragoza, Universidad Nacional Autónoma de México, Mexico City, Mexico; and 2Hospital Gustavo Baz Prada, Instituto de Salud del Estado de México, Nezahualcoyotl, Mexico.

Abstract

OBJECTIVE:

The aim of this study was to determine the influence of menopause (hypoestrogenism) as a risk factor for oxidative stress.

METHODS:

We carried out a cross-sectional study with 187 perimenopausal women from Mexico City, including 94 premenopausal (mean ± SD age, 44.9 ± 4.0 y; estrogen, 95.8 ± 65.7 pg/mL; follicle-stimulating hormone, 13.6 ± 16.9 mIU/mL) and 93 postmenopausal (mean ± SD age, 52.5 ± 3.3 y; estrogen, 12.8 ± 6.8 pg/mL; follicle-stimulating hormone, 51.4 ± 26.9 mIU/mL) women. We measured lipoperoxides using a thiobarbituric acid-reacting substance assay, erythrocyte superoxide dismutase and glutathione peroxidase activities, and the total antioxidant status with the Randox kit. An alternative cutoff value for lipoperoxide level of 0.320 μmol/L or higher was defined on the basis of the 90th percentile of young healthy participants. All women answered the Menopause Rating Scale, the Athens Insomnia Scale, and a structured questionnaire about pro-oxidant factors, that is, smoking, consumption of caffeinated and alcoholic beverages, and physical activity. Finally, we measured weight and height and calculated body mass index.

RESULTS:

The lipoperoxide levels were significantly higher in the postmenopausal group than in the premenopausal group (0.357 ± 0.05 vs 0.331 ± 0.05 μmol/L, P = 0.001). Using logistic regression to control pro-oxidant variables, we found that menopause was the main risk factor for oxidative stress (odds ratio, 2.62; 95% CI, 1.35-5.11; P < 0.01). We also found a positive correlation between menopause rating score, insomnia score, and lipoperoxides, and this relationship was most evident in the postmenopausal group (menopause scale, r = 0.327 [P = 0.001]; insomnia scale, r = 0.209 [P < 0.05]).

CONCLUSIONS:

Our findings suggest that the depletion of estrogen in postmenopause could cause oxidative stress in addition to the known symptoms.

Recent human trial shows oxidative stress to be a key player in menopause

Carcinogenesis. 2001 Jun;22(6):905-11.

Analysis of potential biomarkers of estrogen-initiated cancer in the urine of Syrian golden hamsters treated with 4-hydroxyestradiol.

Todorovic R, Devanesan P, Higginbotham S, Zhao J, Gross ML, Rogan EG, Cavalieri EL.

Estrone (E1) and 17beta-estradiol (E2) are metabolized to catechol estrogens (CE), which may be oxidized to semiquinones and quinones (CE-Q). CE-Q can react with glutathione (GSH) and DNA, or be reduced to CE. In particular, CE-3,4-Q react with DNA to form depurinating adducts (N7Gua and N3Ade), which are cleaved from DNA to leave behind apurinic sites. We report the determination of 22 estrogen metabolites, conjugates and adducts in the urine of male Syrian golden hamsters treated with 4-hydroxyestradiol (4-OHE2). After initial purification, urine samples were analyzed by HPLC with multichannel electrochemical detection and by capillary HPLC/tandem mass spectrometry. 4-Hydroxyestrogen-2-cysteine [4-OHE1(E2)-2-Cys] and N-acetylcysteine [4-OHE1(E2)-2-NAcCys] conjugates, as well as the methoxy CE, were identified and quantified by HPLC, whereas the 4-OHE1(E2)-1-N7Gua depurinating adducts and 4-OHE1(E2)-2-SG conjugates could only be identified by the mass spectrometry method. Most of the administered 4-OHE2 was metabolically converted to 4-OHE1. Formation of thioether (GSH, Cys and NAcCys) conjugates and depurinating adducts [4-OHE1(E2)-1-N7Gua] indicates that oxidation of 4-CE to CE-3,4-Q and subsequent reaction with GSH and DNA, respectively, do occur. The major conjugates in the urine were 4-OHE1(E2)-2-NACCYS: The oxidative pathway of 4-OHE1(E2) accounted for approximately twice the level of products compared with those from the methylation pathway. The metabolites and methoxy CE were excreted predominantly (>90%) as glucuronides, whereas the thioether conjugates were not further conjugated. These results provide strong evidence that exposure to 4-OHE1(E2) leads to the formation of E1(E2)-3,4-Q and, subsequently, depurinating DNA adducts. This process is a putative tumor initiating event. The estrogen metabolites, conjugates and adducts can be used as biomarkers for detecting enzymatic oxidation of estrogens to reactive electrophilic metabolites and possible susceptibility to estrogen-induced cancer.

Oxidation of estrogen may be an initiating event

Effect of vitamin E and C supplements on lipid peroxidation and GSH-dependent antioxidant enzyme status in the blood of women consuming oral contraceptives. Abstract | BACKGROUND: Oral contraceptives (OCs) may affect oxidative stressstatus. We aimed to assess whether supplementation with vitamins E and C reduced this OC effect.

STUDY DESIGN: One hundred twenty healthy female individuals were divided into three groups: A, control; B, untreated OCU (OC users); and C, treated OCU (OC users with vitamin E and C supplementation). In all cases, plasma glutathione peroxidase (GPx) and glutathione reductase (GR) activities and malondialdehyde (MDA) level were determined.

RESULTS: Significant increases were found in the plasma MDA level, and activities of GPx and GR in plasma were decreased in Group B compared to the control group. Supplementation with vitamin C and E significantly increased the activity of GPx and GR activity, and reduced plasma MDA levels in Group C (p<.05).

CONCLUSIONS: These data suggest that low-dose OCs, by enhancing the stress oxidative and lipid peroxidation, may represent a potential cardiovascular risk factor, and the use of vitamins E and C may be beneficial in ameliorating this side effect of OCs.

Oral contraceptives increase oxidative stress

This is particularly significant since 4-hydroxyestrone/estradiol was found to be carcinogenic in the male Syrian golden hamster kidney tumor model, whereas, 2-hydroxylated metabolites were without activity.

. Liehr JG, Fang WR, Sirbasku DA, Ari-Ulubelen A. Carcinogenicity of catechol estrogens in Syrian

hamsters. J. Steroid Biochem 1986;24:353–356. [PubMed: 3009986]

(42). Li JJ, Li SA. Estrogen carcinogenesis in Syrian hamster tissues: role of metabolism. Fed. Proc

1987;46:1858–1863. [PubMed: 3030825]

4-OH and carcinogenesis

Finally, we have shown that a major phase I metabolite of both equilenin and equilin (4-OHEN) can act as a complete carcinogen and tumor promoter in vitro, whereas the endogenous catechol estrogen metabolite, 4-hydroxyestrone was much less effective.

Pisha E, Liu X, Constantinou AI, Bolton JL. Evidence that a metabolite of equine estrogens, 4-

hydroxyequilenin induces cellular transformation in vitro. Chem. Res. Toxicol 2001;14:82–90.

[PubMed: 11170511]

4-OHEN

Osteoporosis and Oxidative Stress

Premarin To Quinones

Interestingly, increasing unsaturation in the B ring leads to a change in metabolism from predominately 2-hydroxylation for estrone to mainly 4-hydroxylation for equilin and exclusively 4-hydroxylation for equilenin. Sarabia SF, Zhu BT, Kurosawa T, Tohma M, Liehr JG. Mechanism of cytochrome P450-catalyzed

aromatic hydroxylation of estrogens. Chem. Res. Toxicol 1997;10:767–771. [PubMed: 9250410]

(56). Zhang F, Chen Y, Pisha E, Shen L, Xiong Y, van Breemen RB, Bolton JL. The Major Metabolite

of Equilin, 4-Hydroxyequilin, Autoxidizes to an o-Quinone Which Isomerizes to the Potent

Cytotoxin 4-Hydroxyequilenin-o- quinone. Chem Res Toxicol 1999;12:204–213. [PubMed:

10027800]

(57

Metabolites

Similar to what has been reported with endogenous estrogens the 4-hydroxylation pathway is predominately catalyzed by P4501B1/1A1.

Spink DC, Zhang F, Hussain MM, Katz BH, Liu X, Hilker DR, Bolton JL. Metabolism of equilenin

in MCF-7 and MDA-MB-231 human breast cancer cells. Chem. Res. Toxicol 2001;14:572–581.

[PubMed: 11368557]

Bolton and Thatcher Page 11

Chem Res Toxicol. Author manuscript; available in PMC 2009 January 1.

NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

4-OH and 1A1

Arch Gerontol Geriatr. 2006 May-Jun;42(3):289-306. Epub 2006 Jan 26.

Menopause: a review on the role of oxygen stress and favorable effects of dietary antioxidants.

Miquel J, Ramírez-Boscá A, Ramírez-Bosca JV, Alperi JD.

Source:Department of Biotechnology, University of Alicante, San Vicente, Ap. 99, E-03080 Alicante, Spain.

Abstract:Menopause is often accompanied by hot flashes and degenerative processes such as arteriosclerosis and atrophic changes of the skin that suggest an acceleration of aging triggered by estrogen lack. Therefore, hormone replacement therapy (HRT) has been considered the most suitable treatment for the above symptoms and processes. However, because of the possible serious side effects of HRT (especially the increased risk of thrombo-embolic accidents and breast cancer) there is a growing demand for alternative treatments of the symptoms and pathological processes associated with menopause. In agreement with the above, we review research that supports the concept that oxygen stress contributes to menopause and that some of its physiopathological effects may be prevented and/or treated improving the antioxidant defense of menopausic and postmenopausic women. Accordingly, a selection of micronutrients may be useful as a dietary supplement for protection against the decline of physiological functions caused by age-related oxygen stress. Since aging is accompanied by a progressive oxidation of the physiological sulfur pool, we emphasize the role of the vitamins B that help to maintain the GSH/GSSG ratio in its normal reduced state. Nutritional supplements should also include the key antioxidant vitamins C and E, as well as beta-carotene and the mineral micronutrients found in the oxygen radical-detoxifying enzymes glutathione peroxidase and superoxide dismutase. Moreover, the reviewed data suport the concept that other antioxidants such as lipoic acid and the precursors of glutathione thioproline (TP) and l-2-oxothiazolidine-4-carboxylic acid (OTC), as well as the soy isoflavones and the "coantioxidants" of an hydroalcoholic extract of Curcuma longa may help to prevent antioxidant deficiency with resulting protection of mitochondria against premature oxidative damage with loss of ATP synthesis and especialized cellular functions. Therefore, the administration under medical advice of synergistic combinations of some of the above mentioned antioxidants in the diet as well as topically (for skin protection) may have favorable effects on the health and quality of life of women, especially of those who cannot be treated with HR, suffer high levels of oxygen stress, and do not consume a healthy diet that includes five daily rations of fresh fruit and vegetables.

HRT and Antioxidant Response Elements

This could be problematic since 2-hydroxylation of endogenous estrogens is regarded as a benign metabolic pathway whereas 4-hydroxylation could lead to carcinogenic metabolites. It can be reasonably hypothesized that metabolism of equilenin and equilin (and their 17β-hydroxylated metabolites) to 4-OHEN is responsible for carcinogensis.

Liehr JG, Fang WR, Sirbasku DA, Ari-Ulubelen A. Carcinogenicity of catechol estrogens in Syrian

hamsters. J. Steroid Biochem 1986;24:353–356. [PubMed: 3009986]

Li JJ, Li SA. Estrogen carcinogenesis in Syrian hamster tissues: role of metabolism. Fed. Proc

1987;46:1858–1863. [PubMed: 3030825]

Newbold RR, Liehr JG. Induction of uterine adenocar,17β-OHEN represents the major carcinogenic pathway for equine estrogens.

4-OH is deleterious

Maturitas. 2001 Apr 20;38(2):165-70.The effects of hormone replacement therapy on lipid peroxidation and antioxidant status.Ozden S, Dildar K, Kadir YH, Gülizar K.SourceDepartment of Biochemistry, Taksim State Hospital, Istanbul, Turkey.AbstractOBJECTIVE:A number of studies have consistently shown a lower cardiovascular risk in women who received postmenopausal hormone replacement therapy (HRT). The aim of our study was to examine the effects of HRT on lipid peroxidation and antioxidant status, which were likely to be involved in the pathophysiology of atherosclerosis.METHODS:We measured erythrocyte and plasma thiobarbituric acid reactive substances (TBARS) levels as expression of lipid peroxidation-end product malondialdehyde, and also erythrocyte reduced glutathione (GSH) level and glutathione peroxidase (GSH-Px) activity as indicators of the antioxidant status of the 35 postmenopausal women with HRT (mean age: 51.81 +/- 4.57 yr; body mass index (BMI): 26.56 +/- 3.78 kg/m(2)) and 35 postmenopausal women without HRT (mean age: 47.50 +/- 3.64; BMI: 27.42 +/-3.43 kg/m2).RESULTS:In the group with HRT, erythrocyte and plasma TBARS levels were significantly lower than in the group without HRT (P < 0.003 and P < 0.001, respectively). Erythrocyte GSH level and GSH-Px activity was found to be increased significantly in the group with HRT in comparison with the group without HRT (P < 0.001 and P < 0.001, respectively). There was not any correlation between the erythrocyte and plasma TBARS and erythrocyte GSH levels and GSH-Px activity with duration of HRT (mean 3.5+/-1.3 yr).CONCLUSION:Our results show that HRT is beneficial in the protection against oxidative damage and can prevent atherosclerotic complications.

Glutathione Peroxidase

Chem Res Toxicol. 1998 Aug;11(8):917-24.Covalent binding of catechol estrogens to glutathione catalyzed by horseradish peroxidase, lactoperoxidase, or rat liver microsomes.Cao K, Devanesan PD, Ramanathan R, Gross ML, Rogan EG, Cavalieri EL.SourceEppley Institute for Research in Cancer, University of Nebraska Medical Center, Omaha, Nebraska 68198-6805, USA.AbstractOxidation of catechol estrogens (CE) leads to the reactive electrophilic CE quinones. Reaction of CE-3,4-quinones with DNA has been implicated in tumor initiation. One pathway to prevent this reaction is conjugation of CE quinones with glutathione (GSH). Four CE, 4-hydroxy estrone (4-OHE1), 4-hydroxyestradiol (4-OHE2), 2-OHE1, and 2-OHE2, were conjugated with GSH after oxidation catalyzed by horseradish peroxidase (HRP), lactoperoxidase (LP), or rat liver microsomal cytochrome P450. This reaction is a free-radical chain autoxidation that produces very high yields of products. Six mono-GSH conjugates, 4-OHE1(E2)-2-SG, 2-OHE1(E2)-1-SG, and 2-OHE1(E2)-4-SG, and four di-GSH conjugates, 4-OHE1(E2)-1,2-bisSG and 2-OHE1(E2)-1,4-bisSG, were identified and quantified. These di-GSH conjugates were also obtained quantitatively from oxidation of mono-GSH conjugates by the same enzymes. HRP and LP gave very similar product profiles. Phenobarbital- and 3-methylcholanthrene-induced microsomes with either NADPH or cumene hydroperoxide as cofactor oxidized 4-OHE2 to form similar amounts of GSH conjugates. Enzymatic oxidation of 2-OHE1(E2) in the presence of GSH produced more 2-OHE1(E2)-4-SG than the 1-isomer. This contrasts with the direct reaction of E1(E2)-2,3-Q and GSH, in which the 1-isomer is formed more abundantly than the 4-isomer (Cao, K., Devanesan, P. D., Ramanathan, R., Gross, M. L., Rogan, E. G., and Cavalieri, E. L. (1998) Chem. Res. Toxicol. 11, 909-916). Competitive enzymatic oxidation of equimolar 4-OHE2 and 2-OHE2 in the presence of an equimolar amount of GSH yielded more 2-OHE2 conjugates than 4-OHE2 conjugates, despite E2-3,4-Q being more reactive with GSH than E2-2,3-Q. These results suggest that 2-OHE2 is a better substrate than 4-OHE2 in the catalytic oxidation to quinones, despite the greater reactivity of E2-3,4-Q, compared to E2-2,3-Q, with GSH.

Glutathione clears 4-OH to 2-OH

Methionine, NAC, Taurine, Sulfur Sulforaphane, Glucosinolates, DIM, Curcumin, Milk Thistle,

Resveratrol Oral acetylated glutathione Topical glutathione ALA, Selenium,

Low Glutathione

Critical role of oxidative stress in estrogen-induced carcinogenesisPNAS April 1, 2003, vol. 100, no. 7 p. 3913Hari K. Bhat*†, Gloria Calaf‡, Tom K. Hei*‡, Theresa Loya§, andJaydutt V. Vadgama¶

Mechanisms of estrogen-induced tumorigenesis in the target organ are not well understood. It has been suggested that oxidative stress resulting from metabolic activation of carcinogenic estrogens plays a critical role in estrogen-induced carcinogenesis. We tested this hypothesis by using an estrogen-induced hamster renal tumor model, a well established animal model of hormonal carcinogenesis. Hamsters were implanted with 17β-estradiol (βE2), 17α-estradiol (αE2), 17α-ethinylestradiol (αEE), menadione, a combination of αE2 and αEE, or a combination of αEE and menadione for 7 months. The group treated with βE2 developed target organ specific kidney tumors. The kidneys of hamsters treated with αE2, αEE, or menadione alone did not show any gross evidence of tumor. Kidneys of hamsters treated with a combination of αE2 and αEE showed early signs of proliferation in the interstitial cells. Kidneys of hamsters treated with a combination of menadione and αEE showed foci of tumor with congested tubules and atrophic glomeruli. βE2-treated tumor-bearing kidneys showed >2-fold increase in 8-iso-prostaglandin F2α (8-iso-PGF2α) levels compared with untreated controls. Kidneys of hamsters treated with a combination of menadione and αEE showed increased 8-iso-PGF2α levels compared with untreated controls, whereas no increase in 8-iso-PGF2α was detected in kidneys of αEE-treated group. A chemical known to produce oxidative stress or a potent estrogen with poor ability to produce oxidative stress, were nontumorigenic in hamsters, when given as single agents, but induced renal tumors, when given together. Thus, these data provide evidence that oxidant stress plays a crucial role in estrogen-induced carcinogenesis.

Oxidative stress and estrogen metabolism, F2-isoprostane increases with increase risk of breast cancer

High F2-Isoprostane

Omega 3 fatty acidsVitamin EGreen Tea Remove heavy metals

CoQ10Glutathione therapies (above)Vitamin CSupport Methylation

Arachidonic acid/F2-Isoprostane

F2-Isoprostane

Correlates with neuroprostanes (DHA, and charged DHA does not incorporate into neurons)

Most reliable marker of oxidative stress in vivo Can lead to inactivation of membrane-bound

receptors or enzymes Decreases membrane fluidity Exists as an oxidative fat in plaques Extensive literature correlating it with

neurologic, cardiovascular and carcinogenic condtions

Isoprostanes: markers and mediators of oxidative stress PAOLO MONTUSCHI, PETER J. BARNES* and L. JACKSON ROBERTS, II†,

(The FASEB Journal. 2004;18:1791-1800.)© 2004 FASEB 1

F2-Isoprostanes are are chemically stable specific products of peroxidation are formed in vivo are present in detectable amounts in all normal tissues

and biological fluids, thus allowing definition of a normal range

levels increase substantially in animal models of oxidant injury

are unaffected by lipid content in the diet might provide a sensitive biochemical basis in dose-

finding studies with antioxidants have no significant daily variability of urinary

isoprostane concentrations in healthy subjects

Measurement of F2-isoprostanes has several advantages over other quantitative markers of oxidative stress

J Urol. 2011 Jun;185(6):2102-7. Epub 2011 Apr 15.

Oxidative stress measured by urine F2-isoprostane level is associated with prostate cancer.

Barocas DA, Motley S, Cookson MS, Chang SS, Penson DF, Dai Q, Milne G, Roberts LJ 2nd, Morrow J, Concepcion RS, Smith JA Jr, Fowke JH.

Oxidative stress is implicated in prostate cancer by several lines of evidence. We studied the relationship between the level of F2-isoprostanes, a validated biomarker of oxidative stress, and prostate cancer and high grade prostatic intraepithelial neoplasia.

MATERIALS AND METHODS:

This case-control analysis within the Nashville Men's Health Study included men recruited at prostate biopsy. Body morphometrics, health history and urine were collected from more than 2,000 men before biopsy. F2-isoprostanes were measured by gas chromatography/mass spectrometry within an age matched sample of Nashville Men's Health Study participants that included 140 patients with high grade prostatic intraepithelial neoplasia, 160 biopsy negative controls and 200 prostate cancer cases. Multivariable linear and logistic regression was used to determine the associations between F2-isoprostane level, and high grade prostatic intraepithelial neoplasia and prostate cancer.

RESULTS:

Mean patient age was 66.9 years (SD 7.2) and 10.1% were nonwhite. Adjusted geometric mean F2-isoprostane levels were higher in patients with prostate cancer (1.82, 95% CI 1.66-2.00) or high grade prostatic intraepithelial neoplasia (1.82, 95% CI 1.68-1.96) than in controls (1.63, 95% CI 1.49-1.78, p <0.001), but were similar across Gleason scores (p = 0.511). The adjusted odds of high grade prostatic intraepithelial neoplasia and prostate cancer increased with increasing F2-isoprostane quartile (p-trend = 0.015 and 0.047, respectively) and the highest F2-isoprostane quartile was associated with significantly increased odds of prostate cancer (OR 2.44, 95% CI 1.17-5.09, p = 0.017).

CONCLUSIONS:

Pre-diagnosis urine F2-isoprostane level is increased in men with high grade prostatic intraepithelial neoplasia or prostate cancer, suggesting urinary F2-isoprostane provides a biomarker for the role for oxidative stress in prostate carcinogenesis. F2-isoprostanes may also serve to estimate the efficacy of interventions targeting oxidative stress mechanisms in prostate cancer prevention or treatment.

Urinary F2-isoprostane provides a possible biomarker for the role for oxidative stress in prostate carcinogenesis.

Arch Intern Med. 2008 Oct 27;168(19):2146-53. doi: 10.1001/archinte.168.19.2146.Levels of sex steroid and cardiovascular disease measures in premenopausal and hormone-treated women at midlife: implications for the "timing hypothesis".Sowers MR, Randolph J Jr, Jannausch M, Lasley B, Jackson E, McConnell D.SourceDepartment of Epidemiology, School of Public Health, University of Michigan, Ann Arbor, MI 48109, USA.AbstractBACKGROUND:The "timing hypothesis," in addressing findings from the Women's Health Initiative trial, suggests that hormone therapy (HT) should be initiated within 6 years of the menopause transition to extend a favorable estrogenic environment after menopause.METHODS:We compared sex steroid and cardiovascular profiles at the 5-year follow-up visit in a community-based, longitudinal study of the menopause transition (Study of Women's Health Across the Nation). Women aged 47 to 57 years were in 1 of 4 groups: premenopausal, women using conjugated equine estrogen with or without progestin, or postmenopausal (<5 years) without HT use. Cardiovascular assays included low-density lipoprotein cholesterol, oxidized low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, triglycerides, apolipoproteins A-I and B, F(2a)-isoprostanes, C-reactive protein, and lipoprotein (a)-1. Sex steroid assays were performed for estradiol, estrogen receptor ligand load, 2-hydroxyestrone, 16alpha-hydroxyestrone, total testosterone, and sex hormone-binding globulin.RESULTS:Users of HT had 50% higher levels of sex hormone-binding globulin (P < .001 for both HT groups), which limits binding of sex steroids to their receptors, and higher excreted estrone metabolites (more than 60%; P < .001 for both HT groups) than premenopausal or postmenopausal women. These findings were, in turn, associated with higher levels of F(2a)-isoprostanes, an oxidative stress measure, than in premenopausal women. The HT users had a more favorable ratio of high-density to low-density lipoprotein cholesterol than did premenopausal or postmenopausal women (P < .01), but higher triglyceride levels (P < .01).CONCLUSION:Although HT users had some more favorable lipid profiles than premenopausal and postmenopausal women, there was evidence of adverse HT effects even in women free of atherosclerosis studied within the approximate 6-year period proposed with the timing hypothesis.

Hormone therapy and F2-Isoprostane

Menopause. 2008 Jul-Aug;15(4 Pt 1):714-7. doi: 10.1097/gme.0b013e31815f8943.Transdermal estradiol reduces F2alpha-isoprostane levels in postmenopausal women.Hermenegildo C, Oviedo PJ, Laguna A, García-Pérez MA, Tarín JJ, Cano A.SourceResearch Foundation, Hospital Clínico Universitario de Valencia, Valencia, Spain.AbstractOBJECTIVE:F2alpha-isoprostanes are considered the most reliable index of in vivo oxidative stress. Given the implication of oxidative stress in the pathogenesis of atherosclerosis, we investigated the effects of hormone therapy on the plasma levels of F2alpha-isoprostanes.DESIGN:Sixty-one healthy postmenopausal women were treated in a randomized trial with estradiol either orally (2 mg/day, 28 women) or transdermally (50 mug/day, 33 women) for 4 weeks. Then women in each group were randomly assigned to oral progestogen, either micronized progesterone (300 mg/day) or medroxyprogesterone acetate (5 mg/day) for 2 additional weeks. Plasma samples were collected before and at the end of each treatment period, either estradiol alone or estradiol plus progestogen. F2alpha-isoprostanes were measured by a commercial enzyme immunoassay.RESULTS:A significant reduction in the levels of F2alpha-isoprostanes was detected only in women receiving transdermal estradiol, alone or in combination with medroxyprogesterone acetate.CONCLUSIONS:Transdermal estradiol alone or associated with medroxyprogesterone acetate decreased plasma levels of F2alpha-isoprostanes. These data elucidate additional details of the beneficial effect of estradiol on oxidative stress, a relevant mechanism in atherogenesis.

Transdermal estradiol reduces F2-Isoprostane

High 8-OHdG

Green Tea CurcuminALAMethylationMetal removal

Glutathione therapies (above)Resveratrol

In support of this mechanism, various free radical toxicities have been reported in hamsters treated with 17β-estradiol including DNA single strand breaks, 8-oxo-dG formation, and chromosomal abnormalities. 

Bolton JL, Trush MA, Penning TM, Dryhurst G, Monks TJ. Role of quinones in toxicology. Chem.

Res. Toxicol 2000;13:135–160. [PubMed: 10725110]

. Roy D, Liehr JG. Estrogen, DNA damage and mutations. Mutat. Res 1999;424:107–115. [PubMed:

10064854]

Nutter LM, Ngo EO, Abul-Hajj YJ. Characterization of DNA damage induced by 3,4-estrone-oquinone in human cells. J. Biol. Chem 1991;266:16380–16386. [PubMed: 1653233]

. Lavigne JA, Goodman JE, Fonong T, Odwin S, He P, Roberts DW, Yager JD. The Effects of

Catechol-O-Methyltransferase Inhibition on Estrogen Metabolite and Oxidative DNA Damage

Levels in Estradiol-treated MCF-7 Cells. Cancer Res 2001;61:7488–7494. [PubMed: 11606384]

Rajapakse N, Butterworth M, Kortenkamp A. Detection of DNA strand breaks and oxidized DNA

bases at the single-cell level resulting from exposure to estradiol and hydroxylated metabolites.

Environ. Mol. Mutagen 2005;45:397–404. [PubMed: 15662657]

Li JJ, Gonzalez A, Banerjee S, Banerjee SK, Li SA. Estrogen carcinogenesis in the hamster kidney:

role of cytotoxicity and cell proliferation. Environ. Health Perspect 1993;5:259–264. [PubMed:

8013417]

Banerjee SK, Banerjee S, Li SA, Li JJ. Induction of chromosome aberrations in Syrian hamste

8-OHdG

PLoS One. 2011;6(11):e27876. doi: 10.1371/journal.pone.0027876. Epub 2011 Nov 29.Raloxifene and desmethylarzoxifene block estrogen-induced malignant transformation of human breast epithelial cells.Kastrati I, Edirisinghe PD, Hemachandra LP, Chandrasena ER, Choi J, Wang YT, Bolton JL, Thatcher GR.SourceDepartment of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Illinois, USA.AbstractThere is association between exposure to estrogens and the development and progression of hormone-dependent gynecological cancers. Chemical carcinogenesis by catechol estrogens derived from oxidative metabolism is thought to contribute to breast cancer, yet exact mechanisms remain elusive. Malignant transformation was studied in MCF-10A human mammary epithelial cells, since estrogens are not proliferative in this cell line. The human and equine estrogen components of estrogen replacement therapy (ERT) and their catechol metabolites were studied, along with the influence of co-administration of selective estrogen receptor modulators (SERMs), raloxifene and desmethyl-arzoxifene (DMA), and histone deacetylase inhibitors. Transformation was induced by human estrogens, and selectively by the 4-OH catechol metabolite, and to a lesser extent by an equine estrogen metabolite. The observed estrogen-induced upregulation of CYP450 1B1 in estrogen receptor negative MCF-10A cells, was compatible with a causal role for 4-OH catechol estrogens, as was attenuated transformation by CYP450 inhibitors. Estrogen-induced malignant transformation was blocked by SERMs correlating with a reduction in formation of nucleobase catechol estrogen (NCE) adducts and formation of 8-oxo-dG. NCE adducts can be formed consequent to DNA abasic site formation, but NCE adducts were also observed on incubation of estrogen quinones with free nucleotides. These results suggest that NCE adducts may be a biomarker for cellular electrophilic stress, which together with 8-oxo-dG as a biomarker of oxidative stress correlate with malignant transformation induced by estrogen oxidative metabolites. The observed attenuation of transformation by SERMs correlated with these biomarkers and may also be of clinical significance in breast cancer chemoprevention.

Catechol estrogen and 8-OHdG as biomarkers of breast cancer risk

Carcinogenesis. 2012 Dec;33(12):2601-10. doi: 10.1093/carcin/bgs300. Epub 2012 Oct 1.Superoxide dismutase 3 is induced by antioxidants, inhibits oxidative DNA damage and is associated with inhibition of estrogen-induced breast cancer.Singh B, Bhat HK.SourceDivision of Pharmacology and Toxicology, School of Pharmacy, University of Missouri-Kansas City, 2464 Charlotte Street, Room 5251, Kansas City, MO 64108, USA.AbstractEpidemiological data and studies in rodent models strongly support the role of estrogens in the development of breast cancers. Oxidative stress has been implicated in this carcinogenic process. We have recently demonstrated that antioxidants vitamin C or butylated hydroxyanisole (BHA) severely inhibit 17β-estradiol (E2)-induced breast tumor development in female ACI rats. The objective of this study was to characterize the mechanism of antioxidant-mediated prevention of breast cancer. Female August Copenhagen Irish (ACI) rats were treated with E2, vitamin C, vitamin C + E2,

BHA and BHA + E2 for up to 8 months. Superoxide dismutase 3 (SOD3) was suppressed in E2-exposed mammary tissues and in mammary tumors of rats treated with E2. This suppression was overcome by co-treatment of rats with E2 and vitamin C or BHA. 8-Hydroxydeoxyguanosine (8-OHdG) levels determined as a marker of oxidative DNA damage were higher in E2-exposed mammary tissues and in mammary tumors compared with age-matched controls. Vitamin C or BHA treatment significantly decreased E2-mediated increase in 8-OHdG levels in the mammary tissues and in MCF-10A cells. Increased DNA damage, colony and mammosphere formation, and migration in SOD3 knocked down MCF-10A cells, and nuclear translocation of SOD3 in vitamin C-treated mammary tissues and in MCF-10A cells suggest protective role of SOD3 against DNA damage and mammary carcinogenesis. Our studies further demonstrate that SOD3 is induced by antioxidants and is regulated through NRF2. SOD3 may thus be an important gene in defense against oxidative stress and in the prevention of estrogen-mediated breast cancer.

Antioxidants, 8-OHdG and breast cancer, Reversal of the biomarker is meaningful

Cancer Lett. 2000 Apr 3;151(1):87-95.Increased formation of oxidative DNA damage, 8-hydroxy-2'-deoxyguanosine, in human breast cancer tissue and its relationship to GSTP1 and COMT genotypes.Matsui A, Ikeda T, Enomoto K, Hosoda K, Nakashima H, Omae K, Watanabe M, Hibi T, Kitajima M.SourceDepartment of Surgery, Keio University School of Medicine, Tokyo, Japan. matsui@mc.med.keio.ac.jpAbstractReactive oxygen species (ROS) induced damage to DNA plays a major role in carcinogenesis. In order to estimate the level of oxidative damage and its role in breast cancer, 8-hydroxy-2'-deoxyguanosine (8-OHdG) was determined in DNA isolated from human breast tissue. Furthermore, we investigated whether polymorphisms in genes for enzymes involved in generation and elimination of ROS had any association with the level of 8-OHdGin breast tissue. In this study, the level of 8-OHdG in DNA was measured by the high performance liquid chromatography-electrochemical detector (HPLC-ECD) method. Genotypes of cytochrome P450 (CYP)1A1, glutathione S-transferase (GST)M 1, GSTP1 and catechol O-methyltransferase (COMT) were determined by PCR-based restriction fragment length polymorphism analysis. A total of 61 Japanese patients were included in the study. The mean level of 8-OHdG in DNA of breast cancer tissues was 2.07 +/- 0.95 per 10(5) dG residues, while the mean level of 8-OHdG in DNA of non-cancerous breast tissues was 1.34 +/- 0.46 per 10(5) dG residues. The 8-OHdG levels in DNA of breast cancer tissues were significantly higher than those of their corresponding non-cancerous breast tissues (P < 0.0001). There was negative correlation between the clinical stage and the mean level of 8-OHdG in DNA of breast cancer tissues. Furthermore, patients with genotype of high GSTP1 activity had lower level of 8-OHdG in DNA ofbreast cancer tissues than others. On the contrary, the mean level of 8-OHdG in DNA of breast cancer tissues was higher among patients with genotype of high COMT activity. Our findings support the assumption that cancer cells are more exposed to oxidative stress than adjacent non-cancerous tissue. Genetic polymorphisms in enzymes involved in ROS metabolism may have a role in individual susceptibility to oxidant-relatedbreast disease. At the same time, reduction of oxidative stress is thought to be a very important measure for primary prevention of breast cancer.PMID: 10766427 [PubMed - indexed for MEDLINE]

Oxidation and Methylation

High oxidative stress decreases methylation and increases quinone production

If no shift in 4-OH estrone with 5-MTHF, think oxidative stress

Hormones

Cancer Cell Int. 2010 Nov 24;10:46. doi: 10.1186/1475-2867-10-46.

Loss of glutathione peroxidase 3 expression is correlated with epigenetic mechanisms in endometrial adenocarcinoma.Falck E1, Karlsson S, Carlsson J, Helenius G, Karlsson M, Klinga-Levan K.Author informationAbstract

Glutathione peroxidase 3 (GPX3) is one of the key enzymes in the cellular defense against oxidative stress and the hepatocyte growth factor receptor, (MET) has been suggested to be influenced by the GPX3 gene expression. In a previous microarray study performed by our group, Gpx3 was identified as a potential biomarker for rat endometrial adenocarcinoma (EAC), since the expression was highly downregulated in rat EAC tumors. Herein, we have investigated the mRNA expression and Gpx3 and Met in rat EAC by real time quantitative PCR (qPCR), and themethylation status of Gpx3. In addition we have examined the expression of GPX3 and MET in 30 human EACs of different FIGO grades and 20 benign endometrial tissues. We found that the expression of GPX3 was uniformly down regulated in both rat and human EAC, regardless of tumor grade or histopathological subtype, implying that the down-regulation is an early event in EAC. The rate of Gpx3 promoter methylationreaches 91%, where biallelic methylation was present in 90% of the methylated tumors. The expression of the Met oncogene was slightly upregulated in EACs that showed loss of expression of Gpx3, but no tumor suppressor activity of Gpx3/GPX3 was detected. Preliminary results also suggest that the production of H2O2 is higher in rat endometrial tumors with down-regulated Gpx3 expression. A likely consequence of loss of GPX3 protein function would be a higher amount of ROS in the cancer cell environment. Thus, the results suggest important clinical implications of the GPX3 expression in EAC, both as a molecular biomarker for EAC and as a potential target for therapeutic interventions.

Glutathione Peroxidase and Endometriosis

Eur J Cancer. 1996 Jun;32A(7):1209-14.Prognostic and aetiological relevance of 8-hydroxyguanosine in human breast carcinogenesis.Musarrat J, Arezina-Wilson J, Wani AA.SourceDepartment of Radiology, Ohio State University, Columbus 43210, USA.AbstractIn order to estimate the level of oxidative damage and its role in breast cancer, the promutagenic oxidative lesion, 8-hydroxy-2'-deoxyguanosine (8-OHdG), was determined in DNA isolated from 75 human breast tissue specimens and from normal and transformed human breast cell lines, utilising a newly developed solid-phase immunoslot blot assay. The amount of 8-OHdG was found to be 0.25 +/- 0.03 pmol/microgram in normal breast tissue from reduction mammoplasty, 0.98 +/- 0.174 pmol/microgram in benign tumours and 2.44 +/- 0.49 pmol/microgram DNA in malignant breast tissue with invasive ductal carcinoma. The malignant tissue had a statistically significant 9.76-fold higher level of 8-OHdG than normal tissue (P < 0.001, Mann-Whitney). A statistically significant 12.9-fold (P = 0.004) higher endogenous formation of 8-OHdG was also observed in cultured breast cancercells compared with normal breast epithelial cells. In addition, a significantly elevated level (3.35-fold higher, P < 0.05) of 8-OHdG observed in oestrogen receptor-positive compared with oestrogen-negative malignant tissues, and in breast cancer cell lines (9.3-fold higher, P = 0.007) suggests a positive relationship between 8-OHdG formation and oestrogen responsiveness. The extent of 8-OHdG adducts did not show a discernible correlation with either the age or the smoking status of the patients. These results indicate that the accumulation of 8-OHdG in DNA has a predictive significance for breast cancer risk assessment and is conceivably a major contributor in the development of breast neoplasia.

8-OHdG and Breast Cancer

Mutat Res. 2007 Jul 10;631(1):62-8. Epub 2007 Apr 20.Urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) and genetic polymorphisms in breast cancer patients.Kuo HW, Chou SY, Hu TW, Wu FY, Chen DJ.SourceInstitute of Environmental Health, China Medical University, No. 91, Hsueh-Shin Road, Taichung, Taiwan. wukuo@mail.cmu.edu.twAbstractReactive oxygen species (ROS) causes damage to DNA, but the role of ROS in breast carcinoma is still not clear. The objective of this study was to measure the urinary 8-OHdG levels of breast cancer patients at each stage of carcinogenesis and assess its association with the development of breast cancer. Sixty patients with malignant breast tumors were matched with 60 control subjects of the same ages in this case control study. Urinary 8-OHdG levels were significantly higher among breast cancer patients than among the control subjects, after making adjustments for confounders such as smoking, coffee consumption and use of oral contraceptives. The breast cancer patients were divided into three groups based on the stages of their cancer; urinary 8-OHdG levels decreased with each stage of breast carcinoma. Using multiple regression and logistic models adjusted for other covariates, urinary 8-OHdG levels significantly correlated with the development of breast cancer. However, it was found that breast cancer was not significantly influenced by CYP1A1, CYP1M1 or NAT2 polymorphisms. In conclusion, it was found that oxygen radical generation occurred within carcinoma cells, but the role of polymorphism of specific genes in the development of breast cancer should be evaluated.

8-OHdG and breast cancer

Transl Res. 2012 Dec;160(6):411-8. doi: 10.1016/j.trsl.2012.07.005. Epub 2012 Aug 10.Oxidative damage markers as possible discriminatory biomarkers in breast carcinoma.Pande D, Negi R, Karki K, Khanna S, Khanna RS, Khanna HD.SourceDepartment of Biophysics, Banaras Hindu University, Varanasi, India.AbstractThe study was designed to evaluate the markers of oxidative damage and to establish their diagnostic utility in breast carcinoma patients. Levels of 8-hydroxy-2-deoxyguanosine (8-OH-dG), protein carbonyl (PC), and malondialdehyde (MDA) along with total antioxidant status (TAS) were measured inbreast carcinoma patients and controls. Receiver operating characteristic (ROC) analysis was done to study the diagnostic potential of the oxidative damage markers. Significant increases in oxidative damage markers were observed in breast carcinoma patients compared with the normal controls, which were accompanied by significant decrease in TAS. The logistic regression analysis revealed higher levels of oxidative stress marker and reduced level of TAS were significantly associated with breast cancer. ROC curves analysis demonstrates that 8-OHdG and PC are better indicators for distinguishing cancer patients from controls, followed by MDA and TAS. Our results indicate increased oxidative damage is associated with malignancy in breast cancer patients. High accuracy of oxidative stress markers in indicating cancer presence can be used as discriminatory makers for efficient diagnosis.

ROS and breast cancer

Oncol Res. 2009;17(10):483-93.Evaluation of molecular markers in a rat model of mammary carcinogenesis.Vinothini G, Murugan RS, Nagini S.SourceDepartment of Biochemistry and Biotechnology, Faculty of Science, Annamalai University, Annamalainagar-608 002, Tamil Nadu, India.AbstractWe sought to evaluate the molecular markers involved in breast tumorigenesis in a rat model that mimics many essential elements of human breast cancer. Female Sprague-Dawley rats were divided into two groups. Animals in group 1 were given a single dose of 7,12-dimethylbenz[a]anthracene (DMBA) (20 mg/rat) dissolved in 1 ml of sesame oil by intragastric intubation. Group 2 animals received basal diet and served as control. We analyzed DMBA-induced changes in the expression of CYP isoforms (CYP1A1 and 1B1) involved in DMBA metabolism, markers of oxidative stress (4HNE, HEL, and 8-OHdG), cell survival and proliferation (PCNA, NF-kappaB-p50, NF-kappaB-p65, GST-P, and p53), apoptosis (Bcl-2, Bax, caspases, Apaf-1, cytochrome C, and Fas), invasion (uPA, MMP-2, MMP-9, TIMP-2, and RECK), and angiogenesis (VEGF, VEGF-R1, HIF-1alpha, and PLGF) by immunohistochemical localization, Western blot, and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. The present study demonstrates increased carcinogen metabolism, oxidative stress, cell proliferation, together with apoptosis evasion, invasion, metastasis, and neovascularization that may confer a selective growth advantage to DMBA-induced mammary tumors. Aberrant expression of multiple molecules in key signaling pathways in Sprague-Dawley rat mammary tumors renders this model as an important tool for monitoring carcinogenic progression and chemointervention.

Biomarkers of breast cancer

Nutr Cancer. 2005;51(2):146-54.Diet and biomarkers of oxidative damage in women previously treated for breast cancer.Thomson CA, Giuliano AR, Shaw JW, Rock CL, Ritenbaugh CK, Hakim IA, Hollenbach KA, Alberts DS, Pierce JP.SourceDepartment of Nutritional Sciences, The University of Arizona, Tucson 85721-0038, USA. cthomson@email.arizona.eduAbstractThis study sought to evaluate the relationship between dietary intake of fat, polyunsaturated fat, saturated fat, arachidonic acid, and selected dietary antioxidants and levels of oxidative damage as measured by urinary levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG) and 8-epi-prostaglandin F2alpha (8-iso-PGF2alpha) in women previously treated for breast cancer. Two hundred two study subjects participating in the Women's Healthy Eating and Living (WHEL) study were included in this ancillary study. Dietary intakes and concentrations of urinary 8-OHdG and 8-iso-PGF2alpha were measured at baseline and 12 mo in the 179 women included in the analytical cohort. Study subjects demonstrated a significant reduction in dietary total, polyunsaturated, and saturated fat intake and a significant increase in vitamins E and C and beta-carotene intake from baseline to 12 mo. Linear mixed-models analysis using baseline and Year 1 data indicated that vitamin E intake was inversely associated with both 8-OHdG and 8-iso-PGF2alpha. 8-Iso-PGF2alpha is increased with increased body mass index (BMI) and polyunsaturated fatty acid (PUFA) intake, indicating an increase in lipid peroxidation with greater BMI and higher PUFA intake. 8-OHdG was inversely related to age but positively related to arachidonic acid, indicating an increase in DNA damage with higher intake of arachidonic acid (meat). The results of this nested case-controlled study provide potential mechanisms by which a high fruit and vegetable, low-fat diet might reduce the recurrence rate of or early-stage breast cancer.

Oxidative markers shifted by better diet which reduced reoccurrence

Food Chem Toxicol. 2002 Aug;40(8):1145-54.Mechanisms of chronic disease causation by nutritional factors and tobacco products and their prevention by tea polyphenols.Weisburger JH, Chung FL.SourceAmerican Health Foundation, One Dana Road, Valhalla, NY 10595, USA. jweisbur@ahf.orgAbstractThe beverage tea, from the top leaves of the plant Camellia sinensis is one of the most widely used beverages in the world, second only to water. Black and green tea have mostly similar actions. The active components are polyphenols, mainly epigallocatechin gallate in green tea, and the tea leaf polyphenol oxidase mediated oxidation to oolong and black tea, yielding other polyphenols, theaflavin and thearubigins. There is 40-50 mg caffeine in a 160-ml cup of tea. The chemopreventive effects of tea depend on: (1) its action as an antioxidant; (2) the specific induction of detoxifying enzymes; (3) its molecular regulatory functions on cellular growth, development and apoptosis; and (4) a selective improvement in the function of the intestinal bacterial flora. The oxidation of LDL cholesterol, associated with a risk for atherosclerosis and heart disease, is inhibited by tea. Many of cancers are caused by lifestyle elements. One is cigarette and tobacco use, leading to cancer in the oral cavity, esophagus and lung, inhibited by tea. Mice administered a tobacco nitrosamine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), developed significantly fewer lung tumors than controls when given green tea or its major polyphenol, epigallocatechin gallate (EGCG). Tea suppressed the formation of 8-hydroxydeoxyguanosine (8-OHdG), a marker of oxidative DNA damage, in the lung DNA of mice given NNK. Gastric cancer, caused by a combination of Helicobacter pylori and salted foods, is lower in tea drinkers. Western nutritionally-linked cancers of the breast, colon, prostate and pancreas can be inhibited by tea. The formation of genotoxic carcinogens for these target organs during the cooking of meats, heterocyclic amines, and their effects were decreased by tea. Tea inhibited the formation of reactive oxygen species and radicals and induced cytochromes P450 1A1, 1A2 and 2B1, and glucuronosyl transferase. The higher formation of glucuronides represents an important mechanism in detoxification. The developmental aspects and growth of cancers through promotion are decreased by tea. The regular use of a widely available, tasty, inexpensive beverage, tea, has displayed valuable preventive properties in chronic human diseases.

8-OHdG and Tea

Carcinogenesis. 2012 Jan;33(1):156-63. doi: 10.1093/carcin/bgr237. Epub 2011 Nov 9.Induction of NAD(P)H-quinone oxidoreductase 1 by antioxidants in female ACI rats is associated with decrease in oxidative DNA damage and inhibition of estrogen-induced breast cancer.Singh B, Bhat NK, Bhat HK.SourceDivision of Pharmacology and Toxicology, School of Pharmacy, University of Missouri-Kansas City, Kansas City, MO 64108, USA.AbstractExact mechanisms underlying the initiation and progression of estrogen-related cancers are not clear. Literature, evidence and our studies strongly support the role of estrogen metabolism-mediated oxidative stress in estrogen-induced breast carcinogenesis. We have recently demonstrated that antioxidants vitamin C and butylated hydroxyanisole (BHA) or estrogen metabolism inhibitor α-naphthoflavone (ANF) inhibit 17β-estradiol (E2)-induced mammary tumorigenesis in female ACI rats. The objective of the current study was to identify the mechanism of antioxidant-mediated protection against E2-induced DNA damage and mammary tumorigenesis. Female ACI rats were treated with E2 in the presence or absence of vitamin C or BHA or ANF for up to 240 days. Nuclear factor erythroid 2-related factor 2 (NRF2) and NAD(P)H-quinone oxidoreductase 1 (NQO1) were suppressed in E2-exposed mammary tissue and in mammary tumors after treatment of rats with E2 for 240 days. This suppression was overcome by co-treatment of rats with E2 and vitamin C or BHA. Time course studies indicate that NQO1 levels tend to increase after 4 months of E2 treatment but decrease on chronic exposure to E2 for 8 months. Vitamin C and BHA significantly increased NQO1 levels after 120 days. 8-Hydroxydeoxyguanosine (8-OHdG) levels were higher in E2-exposed mammary tissue and in mammary tumors compared with age-matched controls. Vitamin C or BHA treatment significantly decreased E2-mediated increase in 8-OHdG levels in the mammary tissue. In vitro studies using silencer RNA confirmed the role of NQO1 in prevention of oxidative DNA damage. Our studies further demonstrate that NQO1 upregulation by antioxidants is mediated through NRF2.

Upregulation of Nrf2 is protective from breast cancer and 8-OHdG helps to monitor this

Enhances Cytoprotection Increases Detoxification Regulates production of Phase 2 Enzymes Enhances stability and turnover of proteins Reduces inflammation Protects against neurodegeneration Anti-tumorigenic Promotes longevity

Nrf2: Master Regulator of CytoProtection

Lewis et al (2010) Integr Comp Biol (May 6, 2010)

Foundation Detox Enhancement SAG Nrf2 Activators Broad Spectrum Antioxidant DIM and SGS Hops Calcium d-glucarate Curcumin and Glutathione cream

Treatment options

Increased Cytoprotection by Nrf2: A Key Contributor to Longevity?

Lewis et al (2010) Integr Comp Biol (May 6, 2010)

Nrf2 Typical anti-oxidant activity is limited in ability and

efficacy to improve / protect against disease

Transcription activators that bind to antioxidant response elements (ARE) in the promoter regions of target genes. Important for the coordinated up-regulation of genes in response to oxidative stress

By activating Nrf2 you can multiply the body's natural antioxidant response to combat inflammation, minimize free radical damage and transport detoxification to new levels.

Nrf2 Activation

Supporting references available upon request

Regulation of Mitochondrial Biogenesis by PGC-1α and Nrf2

Scarpulla RC (2008) Physiol Rev 88:611-638

Sulforaphane Activates Nrf2*

Supporting references available upon request

Natural Antioxidant Activation from Supplementation with Sulforaphane

Turmeric Activates Nrf2

Supporting references available upon request

Curcumin interaction with ROS and Toxic Metabolites

Absorption Benefits of Bioperine

Rats Humans

Source: Shoba G et al. (1998) Planta Medica 64: 353-356

Green Tea Activates Nrf2

Supporting references available upon request

Pterostilbene Activates Nrf2

Supporting references available upon request

Breast Cancer Res Treat. 2010 Nov;124(2):585-91. doi: 10.1007/s10549-010-1023-8. Epub 2010 Jul 10.Inhibition of estrogen signaling activates the NRF2 pathway in breast cancer.Yao Y, Brodie AM, Davidson NE, Kensler TW, Zhou Q.SourceDepartment of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, MD 21201, USA.AbstractExposure to higher levels of estrogen produces genotoxic metabolites that can stimulate mammary tumorigenesis. Induction of NF-E2-related factor 2 (NRF2)-dependent detoxifying enzymes (e.g., NAD(P)H-quinone oxidoreductase 1 (NQO1)) is considered an important mechanism of protection against estrogen-associated carcinogenesis because they would facilitate removal of toxic estrogens. Here, we studied the impact of estrogen-receptor (ER) signaling on NRF2-dependent gene transcription. In luciferase assay experiments using the 5-flanking region of the human NQO1 gene promoter, we observe that ERα ligand-binding domain (LBD) is required for estrogen inhibition of NQO1 promoter activity in estrogen-dependent breast cancer cells. Chromatin immunoprecipitation (ChIP) assay shows that estrogen recruits ERα and a class III histone deacetylase SIRT1 at the NQO1 promoter, leading to inhibition of NQO1 transcription. Inhibition of ERα expression by the antiestrogen shikonin reverses the inhibitory effect of estrogen on NQO1 expression. As a consequence, a chemoprevention study was undertaken to monitor the impact of shikonin

on DNA lesions and tumor growth. Treatment of MCF-7 breast cancer cells with shikonin inhibits estrogen-induced 8-hydroxy-2-deoxyguanosine (8-OHdG), a marker of DNA damage. NQO1 deficiency promotes estrogen-dependent tumor formation, and shikonin inhibits estrogen-dependent tumor growth in an NQO1-dependent manner in MCF-7 xenografts. These results suggest that estrogen-receptor signaling pathway has an inhibitory effect on NRF2-dependent enzymes. Moreover, shikonin reverses the inhibitory effects of estrogen on this pathway and may contribute to breast cancer prevention.

Nrf2 and Breast Cancer

J Investig Med. 2009 Aug;57(6):720-3. doi: 10.231/JIM.0b013e3181adfb5b.

DNA oxidation and antioxidant status in breast cancer.Himmetoglu S, Dincer Y, Ersoy YE, Bayraktar B, Celik V, Akcay T.

SourceDepartment of Biochemistry, and Department of General Surgery, Istanbul University Cerrahpasa Medical Faculty, Istanbul, Turkey.Abstract

PURPOSE:: Oxidant/antioxidant balance has been suggested as an important factor for initiation and progression of cancer. The objective of this study was to determine 8-hydroxydeoxyguanosine (8-OHdG) level as a marker of oxidative DNA damage, glutathione peroxidase (G-Px), and superoxide dismutase (SOD) activities as antioxidant activity, in sera from women with breast cancer.

METHODS:: Forty-nine patients with malign breast tumor were included in the study. Blood samples were collected before the surgical operation. Serum level of 8-OHdG was measured with a competitive enzyme-linked immunusorbent assay kit, SOD, and G-Px activities were measured by spectrophotometric kits.

RESULTS:: 8-Hydroxydeoxyguanosine level and SOD activity were found to be increased in breast cancer group as compared with control group. Glutathione peroxidase activity in the breast cancer group was lower than those in the control group. The ratio of 8-OHdG/G-Px in breast cancerpatients was found to be higher than those in the controls. There were correlations between 8-OHdG and CA19-9 (r = 0.77; P < 0.01); age and G-Px (r = -0.84; P < 0.05) in the breast cancer group.

CONCLUSIONS:: Data show that serum levels of 8-OHdG and SOD activities are higher in patients with breast cancer. Glutathione peroxidase activity is lower in the breast cancer group. Increased ratio of 8-OHdG/G-Px in breast cancer patients is the evidence for impaired oxidant/ antioxidant balance in breast cancer.

Antioxidant Status in Breast Cancer, Serum

https://www.dropbox.com/s/x97kcv4pw01u7z4/Oxidative%20Stress%2C%20the%20Lens%20that%20Bends%20Health%202.pptx?dl=0

Drop Box Link

Chem Res Toxicol. 2008 Jan;21(1):93-101. Epub 2007 Dec 4.Potential mechanisms of estrogen quinone carcinogenesis.Bolton JL, Thatcher GR.

There is a clear association between the excessive exposure to estrogens and the development of cancer in hormone-sensitive tissues (breast, endometrium). It has become clear that there are likely multiple overlapping mechanisms of estrogen carcinogenesis. One major pathway is the extensively studied hormonal pathway, by which estrogen stimulates cell proliferation through nuclear estrogen receptor (ER)-mediated signaling, thus resulting in an increased risk of genomic mutations during DNA replication. A similar "nongenomic pathway", potentially involving newly discovered membrane-associated ERs, also appears to regulate extranuclear estrogen signaling pathways. This perspective is focused on a third pathway involving the metabolism of estrogens to catechols mediated by cytochrome P450 and further oxidation of these catechols to estrogen o-quinones. Oxidative enzymes, metal ions, and in some cases molecular oxygen can catalyze o-quinone formation, so that these electrophilic/redox-active quinones can cause damage within cells by alkylation and/or oxidation of cellular proteins and DNA in many tissues. It appears that the endogenous estrogen quinones primarily form unstable N3-adenine or N7-guanine DNA adducts, ultimately resulting in mutagenic apurinic sites. In contrast, equine estrogen quinones, formed from estrogens present in popular hormone replacement therapy prescriptions, generate a variety of DNA lesions, including bulky stable adducts, apurinic sites, DNA strand cleavage, and oxidation of DNA bases. DNA damage induced by these equine quinones is significantly increased in cells containing ERs, leading us to hypothesize a mechanism involving ER binding/alkylation by the catchol/quinone, resulting in a "Trojan horse". The "Trojan horse" carries the highly redox-active catechol to estrogen -sensitive genes, where high amounts of reactive oxygen species are generated, causing selective DNA damage. Our data further suggest that other key protein targets for estrogen o-quinones could be redox-sensitive enzymes (i.e, GST P1-1, QR). These proteins are involved in stress response cascades that are known to contribute to the regulation of cell proliferation and apoptosis. Finally, it has been shown that catechol estrogens can transform breast epithelial cells into a tumorigenic phenotype and that these transformed cells had differential gene expression of several genes involved in oxidative stress. Given the direct link between excessive exposure to estrogens, metabolism of estrogens, and increased risk of breast cancer, it is crucial that factors that affect the formation, reactivity, and cellular targets of estrogen quinoids be thoroughly explored.

Equine estrogens are oxidative through catechol metabolites

A firm link between female reproductive history and increased risk of developing cancer in the breast and endometrium has been established from epidemiological studies (1-4). The longer women are exposed to estrogens, either through early menarche and late menopause and/or through estrogen replacement therapy, the higher is the risk of developing certain hormone-dependent cancers. It used to be thought that the purported benefits of estrogen replacement therapy, which included the relief of menopausal symptoms, decrease in coronary heart disease, osteoporosis, stroke, and Alzheimer’s disease, justified the use of long-term estrogen replacement therapy. However, the release of the initial results from the Women’s Health Initiative Study in July 2002 cast serious doubt on this paradigm for the treatment of post-menopausal women (5). The estrogen plus progestin arm was halted three years early due to significant increases in breast cancer, coronary heart disease, stroke, and pulmonary embolism, with more recent data suggesting an increase in vascular dementia in women over 65 on estrogen replacement therapy (6). In 2004, the estrogen arm was halted because of increased incidence of stroke (7). A recent analysis of data from the National Cancer Institute’s Surveillance, Epidemiology, and End Results (SEER) registries showed that age-adjusted incidence rate of breast cancer fell sharply (6.7%) in 2003 compared to 2002, which seemed to be related to the drop in the use of HRT (8). Finally, a reanalysis of nine prospective studies has shown that exposure to estrogens is associated with an increase in breast cancer risk with evidence of a dose-response relationship (9). These troubling findings highlight the urgent need for a full understanding of all the deleterious effects of estrogens including their potential to initiate and/or promote the carcinogenic process.

Estrogen Increases Breast Cancer Risk

(1). Liehr JG. Genotoxic effects of estrogens. Mutation Res 1990;238:269–276. [PubMed: 2160607]

(2). Yager JD, Davidson NE. Estrogen carcinogenesis in breast cancer. N. Engl. J. Med 2006;354:270–

282. [PubMed: 16421368]

(3). Colditz GA. Relationship between estrogen levels, use of hormone replacement therapy, and breast

cancer. J. Natl. Cancer Inst 1998;90:814–823. [PubMed: 9625169]

Bolton and Thatcher Page 8

Chem Res Toxicol. Author manuscript; available in PMC 2009 January 1.

NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author Manuscript

(4). Feigelson HS, Henderson BE. Estrogens and breast cancer. Carcinogenesis 1996;17:2279–2284.

[PubMed: 8968038]

(5). Rossouw JE, Anderson GL, Prentice RL, LaCroix AZ, Kooperberg C, Stefanick ML, Jackson RD,

Beresford SA, Howard BV, Johnson KC, Kotchen JM, Ockene J. Risks and benefits of estrogen

plus progestin in healthy postmenopausal women: principal results From the Women’s Health

Initiative randomized controlled trial. Jama 2002;288:321–333. [PubMed: 12117397]

(6). Shumaker SA, Legault C, Rapp SR, Thal L, Wallace RB, Ockene JK, Hendrix SL, Jones BN, Assaf

AR, Jackson RD, Kotchen JM, Wassertheil-Smoller S, Wactawski-Wende J. Estrogen plus

progestin and the incidence of dementia and mild cognitive impairment in postmenopausal women.

The Women’s Health Initiative Memory Study: A Randomized controlled trial. JAMA

2003;289:2651–2662. [PubMed: 12771112]

(7). Brass LM. Hormone replacement therapy and stroke: clinical trials review. Stroke 2004;35:2644–

2647. [PubMed: 15459443]

(8). Ravdin PM, Cronin KA, Howlader N, Berg CD, Chlebowski RT, Feuer EJ, Edwards BK, Berry DA.

The decrease in breast-cancer incidence in 2003 in the United States. N. Engl. J. Med

2007;356:1670–1674. [PubMed: 17442911]

(9). Key T, Appleby P, Barnes I, Reeves G. Endogenous sex hormones and breast cancer in

postmenopausal women: rea

References

In women, significantly higher amounts of GSH conjugates resulting from reaction of GSH with the 4-OHE/E2-o-quinones were detected in the non-tumor tissue from women with breast cancer compared to women without the disease.

Rogan EG, Badawi AF, Devanesan PD, Meza JL, Edney JA, West WW, Higginbotham SM,

Cavalieri EL. Relative imbalances in estrogen metabolism and conjugation in breast tissue of

women with carcinoma: potential biomarkers of susceptibility to cancer. Carcinogenesis

2003;24:697–702. [PubMed: 12727798

Glutathione and Adducts

In addition, estrogen 4-hydroxylase levels (P4501B1 and 1A1) had higher expression in breast tissue of women with breast cancer, whereas expression of protective enzymes was lower.

Singh S, Chakravarti D, Edney JA, Hollins RR, Johnson PJ, West WW, Higginbotham SM, Cavalieri

EL, Rogan EG. Relative imbalances in the expression of estrogen-metabolizing enzymes in the

breast tissue

Increase 4-hydroxylase in breast cancer tissue

Finally, epidemiology studies have suggested a link between genetic polymorphism in estrogen 4-hydroxylases and a risk for developing breast cancer. These data suggest that estrogen metabolites are likely contributors to the development of cancer.

Zheng W, Xie DW, Jin F, Cheng JR, Dai Q, Wen WQ, Shu XO, Gao YT. Genetic polymorphism

of cytochrome P450-1B1 and risk of breast cancer. Cancer Epidemiol. Biomarkers Prev

2000;9:147–150. [PubMed: 10698474]

Kisselev P, Schunck WH, Roots I, Schwarz D. Association of CYP1A1 polymorphisms with

differential metabolic activation of 17beta-estradiol and estrone. Cancer Res 2005;65:2972–2978.

[PubMed: 15805301]

Wen W, Ren Z, Shu XO, Cai Q, Ye C, Gao YT, Zheng W. Expression of cytochrome P450 1B1

and catechol-O-methyltransferase in breast tissue and their associations with breast cancer risk.

Cancer Epidemiol. Biomarkers Prev 2007;16:917–920. [PubMed: 17507616]

Epidemiologic studies and 4-hydroxylases

Most of the epidemiology studies on hormone replacement therapy and cancer risk have investigated the association with use of Premarin® (conjugated equine estrogens, Wyeth-Ayerest) or Prempro® (conjugated equine estrogens plus progestin). Both Premarin® and Prempro® are currently the most popular forms of HRT, widely prescribed in North America.

Since Premarin® was approved by the Food and Drug Administration in the 1940s, very little is known about the metabolism and potential toxic metabolites that could be produced from the various equine estrogens, which make up approximately 50% of the estrogens in Premarin® .

It is known that treating hamsters for 9 months with either estrone, equilin + equilenin, or sulfatase-treated Premarin®, resulted in 100% kidney tumor incidences and abundant tumor foci.

Furthermore, in a small clinical trial of 596 postmenopausal women, a significant increase in endometrial hyperplasia was found in those women receiving a daily dose of 0.625 mg of Premarin® ). Finally, we have shown that a major phase I metabolite of both equilenin and equilin (4-OHEN) can act as a complete carcinogen and tumor promoter in vitro, whereas the endogenous catechol estrogen metabolite, 4-hydroxyestrone was much less effective .

As a result, it is quite possible that the B-ring unsaturated equine estrogens have very different biological properties in vivo compared to the endogenous catechol estrogens.

Premarin

(52). Wysowski DK, Governale LA. Use of menopausal hormones in the United States, 1992 throughJune, 2003. Pharmacoepidemiol. Drug Saf 2005;14:171–176. [PubMed: 15386701](53). Purdy RH, Moore PH, Williams MC, Goldzheher HW, Paul SM. Relative rates of 2- and 4-hydroxyestrogen synthesis are dependent on both substrate and tissue. FEBS Lett 1982;138:40–44.[PubMed: 6279439](54). Li JJ, Li SA, Oberley TD, Parsons JA. Carcinogenic activities of various steroidal and nonsteroidalestrogens in the hamster kidney: relation to hormonal activity and cell proliferation. Cancer Res1995;55:4347–4351. [PubMed: 7671246](55). Sarabia SF, Zhu BT, Kurosawa T, Tohma M, Liehr JG. Mechanism of cytochrome P450-catalyzedaromatic hydroxylation of estrogens. Chem. Res. Toxicol 1997;10:767–771. [PubMed: 9250410](56). Zhang F, Chen Y, Pisha E, Shen L, Xiong Y, van Breemen RB, Bolton JL. The Major Metaboliteof Equilin, 4-Hydroxyequilin, Autoxidizes to an o-Quinone Which Isomerizes to the PotentCytotoxin 4-Hydroxyequilenin-o- quinone. Chem Res Toxicol 1999;12:204–213. [PubMed:10027800](57). Bhavnani BR. Pharmacokinetics and pharmacodynamics of conjugated equine estrogens: Chemistryand metabolism. Proc. Soc. Exp. Biol. Med 1998;217:6–16. [PubMed: 9421201](58). Judd HL, Mebane-Sims I, Legault C, Wasilauskas C, Johnson S, Merino M, Barrett-Connor B,Trabal J. Effects of hormone replacement therapy on endometrial histology in postmenopausalwomen. JAMA 1996;275:370–375. [PubMed: 8569016](59). Pisha E, Liu X, Constantinou AI, Bolton JL. Evidence that a metabolite of equine estrogens, 4-hydroxyequilenin induces cellular transformation in vitro. Chem. Res. Toxicol 2001;14:82–90.[PubMed: 11170511](60). Prokai-Tatrai K, Prokai L. Impact of metabolism o

References

In addition, extraction of mammary tissue DNA, hydrolysis to deoxynucleosides, and analysis by LC-MS-MS showed the formation of 8-oxo-dG as well as 8-oxo-dA. Finally, a recent study evaluated the potential of HRT to induce DNA damage in peripheral blood leukocytes of postmenopausal women using the comet assay.

Significant increases in DNA damage were observed between women receiving 0.625 mg/day conjugated equine estrogens or conjugated equine estrogens plus medroxyprogesteron acetate as compared to the control group that had never received HRT.

Increase in ROS with Equine

Finally, the excessive production of reactive oxygen species in breast cancer tissue has been linked to metastasis of tumors in women with breast cancer. These and other data are evidence for a mechanism of estrogen-induced tumor initiation/promotion by redox cycling of estrogen metabolites generating reactive oxygen species, which damage DNA.

Malins DC, Polissar NL, Gunselman SJ. Progession of human breast cancers to the metastatic state is linked to hydroxyl radical-induced DNA damage. Proc. Natl. Acad. Sci. USA 1996;93:2557–2563. [PubMed: 8637913]

Malins DC, Anderson KM, Jaruga P, Ramsey CR, Gilman NK, Green VM, Rostad SW, Emerman

JT, Dizdaroglu M. Oxidative changes in the DNA of stroma and epithelium from the female breast:

potential implications for breast cancer. Cell Cycle 2006;5:1629–1632. [PubMed: 16880742]

Karihtala P, Soini Y. Reactive oxygen species and antioxidant mechanisms in humans

Increase Risk of Mets with Ox Stress

Simultaneously, oxidative DNA damage, such as 7,8-dihydro-8-oxodeoxyguanine (8-oxodG), is also generated by reactive oxygen species through redox cycling between the o-quinone of 4-OHEN and its semiquinone radicals.

Chen Y, Shen L, Zhang F, Lau SS, van Breemen RB, Nikolic D, Bolton JL. The equine estrogen metabolite 4-hydroxyequilenin causes DNA single-strand breaks and oxidation of DNA bases in vitro. Chem. Res. Toxicol. 1998;11:1105–1111. [PubMed]

Quinones and 8-OHdG

Adducts

The rank order of DNA adduct formation which correlated with cellular transformation was 4-OHE2 > 2-OHE2 > E2. Finally, stable bulky adducts of 4-OHE1 and 4-OHE2 corresponding to alkylation of guanine have been detected in human breast tumor tissue.

Embrechts J, Lemiere F, Dongen WV, Esmans EL, Buytaert P, van Marck E, Kockx M, Makar A. Detection of estrogen DNA-adducts in human breast tumor tissue and healthy tissue by combined nano LC-nano ES tandem mass spectrometry. J. Am. Soc. Mass Spectrom. 2003;14:482–491. [PubMed]

Rank of order of DNA adduct formation

For the major equine estrogens (equilin and equilenin and 17β-ol derivatives) the data strongly suggests that the majority of DNA damage results from reactions of 4-OHEN-o-quinone through a combination of oxidative damage (i.e., single strand cleavage and oxidation of DNA bases) and through generation of apurinic sites as well as stable bulky cyclic adducts.

Bolton JL. Quinoids, quinoid radicals, and phenoxyl radicals formed from estrogens and

antiestrogens. Toxicology 2002;177:55–65. [PubMed: 12126795]

Equine estrogen and 4-OH

Finally, the endogenous catechol estrogen metabolite 4-hydroxyestrone was considerably less effective at inducing DNA damage in breast cancer cell lines as compared to 4-OHEN. Our data suggest that the genotoxic effects of 4-OHEN could be related to its ability to induce DNA damage in hormone sensitive cells in vivo, and these effects may be potentiated by the ER.

Chen Y, Liu X, Pisha E, Constantinou AI, Hua Y, Shen L, van Breemen RB, Elguindi EC, Blond

SY, Zhang F, Bolton JL. A metabolite of equine estrogens, 4-hydroxyequilenin, induces DNA

damage and apoptosis in breast cancer cell lines. Chem. Res. Toxicol 2000;13:342–350. [PubMed:

10813650]

4-OH to 4-OHEN

PLoS One. 2011;6(9):e25125. doi: 10.1371/journal.pone.0025125. Epub 2011 Sep 26.

Partial inhibition of estrogen-induced mammary carcinogenesis in rats by tamoxifen: balance between oxidant stress and estrogen responsiveness.

Singh B, Bhat NK, Bhat HK.

Source

Division of Pharmacology and Toxicology, School of Pharmacy, University of Missouri-Kansas City, Kansas City, Missouri, United States of America.

Abstract

Epidemiological and experimental evidences strongly support the role of estrogens in breast tumor development. Both estrogen receptor (ER)-dependent and ER-independent mechanisms are implicated in estrogen-induced breast carcinogenesis. Tamoxifen, a selective estrogen receptor modulator is widely used as chemoprotectant in human breast cancer. It binds to ERs and interferes with normal binding of estrogen to ERs. In the present study, we examined the effect of long-term tamoxifen treatment in the prevention of estrogen-induced breast cancer. Female ACI rats were treated with 17β-estradiol (E2), tamoxifen or with a combination of E2 and tamoxifen for eight months. Tissue levels of oxidative stress markers 8-iso-Prostane F(2α) (8-isoPGF(2α)), superoxide dismutase (SOD), glutathione peroxidase (GPx), catalase, and oxidative DNA damage marker 8-hydroxydeoxyguanosine (8-OHdG) were quantified in the mammary tissues of all the treatment groups and compared with age-matched controls. Levels of tamoxifen metabolizing enzymes cytochrome P450s as well as estrogen responsive genes were also quantified. At necropsy, breast tumors were detected in 44% of rats co-treated with tamoxifen+E2. No tumors were detected in the sham or tamoxifen only treatment groups whereas in the E2 only treatment group, the tumor incidence was 82%. Co-treatment with tamoxifen decreased GPx and catalase levels; did not completely inhibit E2-mediated oxidative DNA damage and estrogen-responsive genes monoamine oxygenase B1 (MaoB1) and cell death inducing DFF45 like effector C (Cidec) but differentially affected the levels of tamoxifen metabolizing enzymes. In summary, our studies suggest that although tamoxifen treatment inhibits estrogen-induced breast tumor development and increases the latency of tumor development, it does not completely abrogate breast tumor development in a rat model of estrogen-induced breast cancer. The inability of tamoxifen to completely inhibit E2-induced breast carcinogenesis may be because of increased estrogen-mediated oxidant burden.

Oxidative Stress and Tamoxifen Therapy

J Biol Chem. 2013 Mar 6. [Epub ahead of print]Deleterious cholesterol hydroperoxide trafficking in star protein-expressing MA-10 leydig cells: implications foroxidative stress-impaired steroidogenesis.Korytowski W, Pilat A, Schmitt JC, Girotti AW.SourceMedical College of Wisconsin/Jagiellonian University, United States;AbstractStAR proteins in steroidogenic cells are implicated in the delivery of cholesterol (Ch) from internal or external sources to mitochondria (Mito) for initiation of steroid hormone synthesis. In this study, we tested the hypothesis that under oxidative stress, StAR-mediated trafficking of redox-active cholesterol hydroperoxides (ChOOHs) can result in site-specific Mito damage and dysfunction. Steroidogenic stimulation of mouse MA-10 Leydig cells with dibutyryl-cAMP (db-cAMP) resulted in strong

expression of StarD1 and StarD4 proteins over insignificant levels in non-stimulated controls. During incubation with the ChOOH 7α-OOH in liposomes, stimulated cells took up substantially more hydroperoxide in Mito than controls, with a resulting loss of membrane potential (Ψm) and ability to drive progesterone synthesis. 7α-OOH uptake and Ψm loss were greatly reduced by StarD1 knockdown, thus establishing this proteins role in 7α-OOH delivery. Moreover, 7α-OOH was substantially more toxic to stimulated than non-stimulated cells, the former dying mainly by apoptosis and the latter by necrosis. Importantly, tert-butyl hydroperoxide, which is not a StAR protein ligand, was equally toxic to stimulated and non-stimulated cells. These findings support the notion that like Ch itself, 7α-OOH can be transported to/into Mito of steroidogenic cells by StAR proteins, and therein induce free radical damage which compromises steroid hormone synthesis.

Hormone production and oxidative stress

Endocr J. 2013 Jan 31;60(1):1-13. Epub 2012 Dec 22.Melatonin as a free radical scavenger in the ovarian follicle.Tamura H, Takasaki A, Taketani T, Tanabe M, Kizuka F, Lee L, Tamura I, Maekawa R, Asada H, Yamagata Y, Sugino N.SourceDepartment of Obstetrics and Gynecology, Yamaguchi University Graduate School of Medicine, Ube 755-8505, Japan. hitamura@yamaguchi-u.ac.jpAbstractThis review summarizes new findings related to beneficial effects of melatonin (N-acetyl-5-methoxytryptamine) on reproductive physiology. Recently many researchers have begun to study the local role of melatonin as an antioxidant. We focused on intra-follicular role of melatonin in the ovary. Melatonin, secreted by the pineal gland, is taken up into the follicular fluid from the blood. Reactive oxygen species (ROS) are produced within the follicles, during the ovulatory process. Melatonin reduces oxidative stress as an antioxidant, and contribute to oocyte maturation, embryo development and luteinization of granulosa cells. Our clinical study demonstrated that melatonin treatment for infertile women increases intra-follicular melatonin concentrations, reduces intra-follicular oxidative damage, and elevates fertilization and pregnancy rates. Melatonin treatment also improves progesterone production by corpus luteum in infertile women with luteal phase defect. Melatonin treatment could become a new cure for improving oocyte quality and luteal function in infertile women.

Melatonin and fertility

Theriogenology. 2012 Aug;78(3):620-31. doi: 10.1016/j.theriogenology.2012.03.008. Epub 2012 Apr 26.Molecular mechanisms of a novel selenium-based complementary medicine which confers protection against hyperandrogenism-induced polycystic ovary.Rezvanfar MA, Rezvanfar MA, Ahmadi A, Shojaei-Saadi HA, Baeeri M, Abdollahi M.SourceLaboratory of Toxicology, Department of Toxicology and Pharmacology, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran, Iran.AbstractThe objective was to evaluate ovarian functionality and oxidative response in hyperandrogenism-induced polycystic ovary (PCO) and the protective effects of immunomodulator drug (IMOD), an electromagnetically-treated, selenium-based, herbal medicine. Daily oral administration of letrozole (1 mg/kg) for 21 consecutive days induced ovarian cysts in female rats. An effective dose of IMOD (30 mg/kg per day) was given intraperitoneally for 21 days. Biomarkers of ovarian function, serum concentrations of estradiol, progesterone, testosterone, and ovarian prostaglandin-E (PGE), were analyzed. To determine the role of oxidative stress (OS) in hyperandrogenism-induced PCO, concentrations of cellular lipid peroxidation (LPO), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), peroxynitrite (ONOO), and tumor necrosis factor (TNF)-α as a marker of inflammation and apoptosis were measured in serum and ovaries. Letrozole-induced PCO resulted in significant increases in concentrations of lipid peroxidation and peroxynitrite in serum and ovary, but significantly decreased superoxide dismutase, catalase, and glutathione peroxidase. Serum concentrations of testosterone and TNF-α, and ovarian prostaglandin-E were increased (P < 0.001) in animals with cysts versus control, whereas estradiol and progesterone were decreased (P < 0.01 and P < 0.001, respectively). When compared with controls, letrozole induced irregular cycles and PCO characterized by a high incidence of subcapsular ovarian cysts with a diminished granulosa cell layer, luteinized granulosa cells in the cyst wall, significantly more atretic preantral and antral follicles, and absence of CL. There were almost no intact primary, secondary, and tertiary follicles in PCO rats. All end points assessed were significantly improved by IMOD and reached close to normal levels. In conclusion, the present study provided evidence that toxic free radicals and TNF-α were involved in the pathogenesis of PCO; furthermore, IMOD prevented ovarian histopathologic, endocrine, and biochemical alterations induced by hyperandrogenism.

Oxidative stress and PCOS

Initial Treatment: Omega 3, Vitamin D, ALA Testing: Oxidative stress

MS Patient/Hashimotos

Patient Name: S R

Advanced Oxidative Stress Profile: 2800

Patient Results Reference RangeGlutathione 1.5 1.0-14.0 µM

Antioxidant (Intracellular)

Lipid PeroxidationPatient Results Reference Range

TBARS/MDA 17.0 1.0-40.0 µM

F2-isoprostane 23.1 1.0-14.4 ng/mL

Patient Results Reference Range8-OHdG 68.0 1.0-13.6 ng/mg**

DNA Damage

Enzyme AssaysPatient Results Reference Range

SOD I 3.0 3.0-33.8 mU/mL

SOD II 32.0 1.0-26.3 mU/mL

Circadian variation in multiple sclerosis of oxidative stress marker of DNA damage. A potential cancer marker?

Kanabrocki EL, Ryan MD, Murray D, Jacobs RW, Wang J, Hurder A, Friedman NC, Siegel G, Eladasari B, Nemchausky BA, Cornelissen G, Halberg F.

Clin Ter. 2006 Mar-Apr;157(2):117-22.

Edward Hines Jr. Medical Center, Hines, IL 60141, USA. Eugene.Kanabrocki@med.va.govOBJECTIVE: To investigate circadian rhythm (CR) of urinary creatinine and 8-hydroxy-2-deoxyguanosine (8-OHdG) in patients with

Multiple Sclerosis (MS) and to present concentrations of this DNA damage marker, 5 years prior to mastectomy, in one MS study subject, and 2 years prior to biopsy confirmed a carcinoma (CA) of the prostate in one non-MS subject.

MATERIALS AND METHODS: Eleven subjects with MS (6 women 36-52 years of age and 5 men 51-68 years) volunteered for this study, carried out at Edward Hines Jr., Medical Center. Subjects were offered a general hospital diet (2400 cal in total/24h) at 16:30h, 07:30h and 13:00h. The dark (sleep) phase of the light-dark cycle extended from 22:30h to 06:30h with brief awakening for sampling at 01:00h, and 04:00h. Urine samples were collected for consecutive 3h spans beginning at 16:00-19:00h and were analyzed for creatinine and 8-OHdG. Twelve men (including 3 with type 2 diabetes) provided 21 profiles according to the same protocol used for comparison. In addition, 10 healthy women provided 24h urine samples. Statistical analysis of data was performed using the Single-Cosinor and Population-Mean Cosinor.

RESULTS: A CR was detected for creatinine in healthy men (p < 0.001) but not for MS patients. Urinary creatinine concentrations were lower in MS women than in healthy women (p = 0.015) and were lower in MS women than in men healthy or with MS (p < 0.001): Women; MS 655 +/- 76; H 1381 +/- 316; Men, MS 1830 +/- 285; H 1532 +/- 265 mg/24h vol. A CR was evident in 8-OHdG in MS (p = 0.007) and in non-MS subjects (p < 0.001) with highest values occurring at about 16:45h. The average concentrations of 8-OHdG in MS patients were similar to those in healthy subjects: Women, MS 589 +/- 125; H 794 +/- 318; Men, MS 504 +/- 156; H 591 +/- 134 picomoles/kg bw/24h vol. The 8-OHdG concentrations of a MS patient, later diagnosed with breast cancer, were found to exceed the upper 95% prediction limit in health. An increased 8-OHdG level was also noted in a non-MS subject who 2 years later received a biopsy-confirmed diagnosis of prostate CA.

CONCLUSIONS: Despite the small number of subjects in this study, a statistically significant CR was documented for 8-OHdG in urine of subjects with MS. Interestingly, the increased concentrations of DNA damage marker, the 8-OHdG, 5 years prior to mastectomy and the 2 years prior to affirmative diagnosis of prostate CA, could be the most significant clinical observations of this study. Follow-up studies of a larger population of subjects would, thus, be required to ascertain the predictive validity of such challenging observation.

8-0HdG elevation MS/cancer

Structure-dependent inhibition of gelatinases by dietary antioxidants in rat astrocytes and sera of multiple sclerosis patients.

Neurochem Res. 2011 Mar;36(3):518-27. Epub 2011 Jan 5.

Liuzzi GM, Latronico T, Branà MT, Gramegna P, Coniglio MG, Rossano R, Larocca M, Riccio P.Department of Biochemistry and Molecular Biology, Ernesto Quagliariello, University of Bari, Via Orabona 4,

70126, Bari, Italy. m.g.liuzzi@biologia.uniba.it

We investigated whether polyphenols modulate the expression and activity of the enzymes gelatinases A (MMP-2) and B (MMP-9), involved in the pathogenesis of multiple sclerosis (MS). LPS-activated primary rat astrocytes were treated with the flavonoids quercetin (QRC) and cathechins [green tea extract (GTE)] and the non-flavonoids resveratrol (RSV) and tyrosol/hydroxytyrosol (Oliplus). As assessed by zymography and RT-PCR, RSV and Oliplus, but not QRC and GTE, dose-dependently inhibited the LPS-induced levels and mRNA expression of MMP-2 and MMP-9. By contrast, in cell-free systems direct inhibition of gelatinase activity in MS sera was determined by QRC and GTE, but not by RSV. Oliplus was only partially effective. Our results indicate that the flavonoids and non-flavonoids tested exert their inhibitory effect on MMPs, displaying different mechanisms of action, possibly related to their structure. Therefore, their combined use may represent a powerful tool for the down-regulation of MMPs in the course of MS.

Synergy between Green tea and Resveratrol

Levels of reduced and oxidized coenzyme Q-10 and 8-hydroxy-2'-deoxyguanosine in the CSF of patients with Alzheimer's disease demonstrate that mitochondrial oxidative damage and/or oxidative DNA damage contributes to the neurodegenerative process.

J Neurol. 2010 Mar;257(3):399-404. Epub 2009 Sep 27.

Isobe C, Abe T, Terayama Y.Department of Neurology, Iwate Medical University, 19-1 Uchimaru, Morioka, Iwate, 020-0805,

Japan. chiso@apost.plala.or.jp

To investigate the possibility that mitochondrial oxidative damage, oxidative DNA damage or both contribute to the neurodegenerative process of Alzheimer's disease (AD), we employed high-performance liquid chromatography using an electrochemical detector to measure the concentrations of the reduced and oxidized forms of coenzyme Q-10 (CoQ-10) and 8-hydroxy-2'-deoxyguanosine (8-OHdG) in the cerebrospinal fluid (CSF) of 30 patients with AD and in 30 age-matched controls with no neurological disease. The percentage of oxidized/total CoQ-10 (%CoQ-10) in the CSF of the AD group (78.2 +/- 18.8%) was significantly higher than in the control group (41.3 +/- 10.4%) (P < 0.0001). The concentration of 8-OHdG in the CSF of AD patients was greater than in the CSF of controls (P < 0.0001) and was positively correlated with the duration of illness (r(s) = 0.95, P < 0.0001). The %CoQ-10 was correlated with concentrations of 8-OHdG in the CSF of AD patients (r(s) = 0.66, P < 0.001). The present study suggests that both mitochondrial oxidative damage and oxidative DNA damage play important roles in the pathogenesis of early AD development.

8-OHdG

Increased 8-hydroxy-deoxyguanosine, a marker of oxidative damage to DNA, in major depression and myalgic encephalomyelitis / chronic fatigue syndrome.

Neuro Endocrinol Lett. 2009;30(6):715-22.

Maes M, Mihaylova I, Kubera M, Uytterhoeven M, Vrydags N, Bosmans E.Maes Clinics, Wilrijk - Antwerp, Belgium. crc.mh@telenet.be

BACKGROUND: There is now evidence that major depression and myalgic encephalomyelitis / chronic fatigue syndrome (ME/CFS) are accompanied by partially overlapping pathophysiological mechanisms, i.e. activation of various inflammatory and oxidative & nitrosative (IO&NS) pathways.

OBJECTIVE: The aim of the present study was to examine the urinary excretion of 8-hydroxy-deoxyguanosine (8-OhdG), a marker of oxidative damage to DNA, in depression; ME/CFS; and depression and ME/CFS.

METHODS: Toward this end, morning urine was sampled for the assays of 8-OHdG and creatinine, in 44 patients with ME/CFS; 25 with major depression; 23 with depression and ME/CFS; and 17 normal controls. Severity of fatigue and somatic symptoms was measured by means of the Fibromyalgia and CFS Rating (FF) scale.

RESULTS: We found that 49.0% of the variance in the urinary excretion of 8-OHdG was predicted by the regression on creatinine. Consequently, the urinary 8-OHdG excretion should be expressed as the residualized 8-OHdG values after partialling out the effects of creatinine and not by computing the 8-OHdG / creatinine ratio. We found that the residualized urinary excretion of 8-OHdG (adjusted for creatinine) was significantly higher in patients with depression and ME/CFS than in normal controls and all other patients. In the patient group, there were significant correlations

between the urinary 8-OHdG and the total score on the FF scale and sadness and flu-like malaise.

CONCLUSIONS: The findings show increased oxidatively generated DNA damage in patients with major depression and ME/CFS and, therefore, further extent the role played by IO&NS pathways in the pathophysiology of both disorders.

Since oxidatively damage to DNA is a risk factor for atherosclerosis and neurodegeneration, our results also explain previous findings on increased cardiovascular morbidity in depression and ME/CFS, and neurodegenerative processes in depression.

8-OHdG

Green tea (Camellia sinensis) attenuates nephropathy by downregulating Nox4 NADPH oxidase in diabetic spontaneously hypertensive rats.

Ribaldo PD, Souza DS, Biswas SK, Block K, Lopes de Faria JM, Lopes de Faria JB.Laboratory of Renal Pathophysiology, Nephrology Unit, Faculty of Medical Sciences, State University of

Campinas, Campinas, SP, 13084-971 Brazil.J Nutr. 2009 Jan;139(1):96-100. Epub 2008 Dec 3.

Green tea (GT), through its antioxidant properties, may be useful to treat or prevent human diseases. Because several lines of evidence suggest that oxidative stress contributes to the pathogenesis of diabetic nephropathy, we tested the hypothesis that GT prevents diabetes and hypertension-related renal oxidative stress, attenuating renal injury. Spontaneously hypertensive rats (SHR) with streptozotocin-induced diabetes and nondiabetic SHR were treated daily with tap water or freshly prepared GT (13.3 g/L). After 12 wk, the systolic blood pressure did not differ between treated and untreated nondiabetic or diabetic rats. However, body weight was less (P < 0.05) and glycemia was greater in diabetic SHR rats than in nondiabetic rats. Renal oxidative stress variables such as 8-hydroxy-2'-deoxyguanosine (8-OHdG) and nitrotyrosine expression, NADPH oxidase-dependent superoxide generation, and the expression of renal cortex Nox4 were greater (P < 0.05) in diabetic rats that received water (DW) than in nondiabetic rats that received water (CW). The 8-OHdG and NADPH oxidase-dependent superoxide generation were significantly less in rats treated with GT. Nitrotyrosine and Nox4 expression were significantly less in diabetic rats that received GT (DGT) than in DW. Likewise, the indices of renal injury, albuminuria, and renal expression of collagen IV were significantly greater in DW than in CW. These differences were significantly less in DGT than in DW. GT reestablished the redox state and reduced the indicators of nephropathy without altering glycemia and blood pressure levels in diabetic SHR. These findings suggest that the consumption of GT may ameliorate nephropathy in diabetic hypertensive patients.

Green Tea and Neuropathy

Clinical importance of erythrocyte malondialdehyde levels as a marker for cognitive deterioration in patients with dementia of Alzheimer type: a repeated study in 5-year interval.

Clin Biochem. 2002 Mar;35(2):137-41.

Delibas N, Ozcankaya R, Altuntas I.Suleyman Demirel University, School of Medicine, Department of Biochemistry and Clinical Biochemistry,

Isparta, Turkey. ndelibas@hotmail.com

OBJECTIVES: The aim of the present study was to determine the effects of aging and dementia of the Alzheimer type (DAT) on lipid peroxidation and antioxidant enzyme activities.

DESIGN AND METHODS: Twenty-six patients with DAT were included in the present study. Group I: 26 patients diagnosed as DAT and studied 5 yr ago. Group II: This group consisted of the same patients as Group I at the present time. Activities of CuZn superoxide dismutase (CuZn SOD) and glutathione peroxidase (GSH-Px) and malondialdehyde (MDA) concentrations of these 26 subjects were measured and mini mental state examination (MMSE), brief psychiatric rating scale (BPRS), Hamilton depression rating scale (HDRS) were applied.

RESULTS: The results revealed that 26 dementia patients had worsened cognitive symptoms and significantly increased CuZn SOD and MDA levels and decreased GSH-Px levels after 5 yr. Significant correlation was found between age and CuZn SOD (r: +0.406, p: 0.034), and between MMSE and MDA (r: -0.411, p: 0.032).

CONCLUSIONS: We can conclude that MDA, CuZn SOD, and GSH-Px were significantly affected in the patients with Alzheimer disease. The most striking finding of this study is the significant correlation between MMSE and MDA in patients with DAT.

SOD I and dementia

Mech Ageing Dev. 2007 Nov-Dec;128(11-12):706-16. Epub 2007 Nov 4.Reduced mitochondrial SOD displays mortality characteristics reminiscent of natural

aging.Paul A, Belton A, Nag S, Martin I, Grotewiel MS, Duttaroy A.Biology Department, Howard University, NW, Washington, DC 20059, USA.

Manganese superoxide dismutase (MnSOD or SOD2) is a key mitochondrial enzymatic antioxidant. Arguably the most striking phenotype associated with complete loss of SOD2 in flies and mice is shortened life span. To further explore the role of SOD2 in protecting animals from aging and age-associated pathology, we generated a unique collection of Drosophila mutants that progressively reduce SOD2 expression and function. Mitochondrial aconitase activity was substantially reduced in the Sod2 mutants, suggesting that SOD2 normally ensures the functional capacity of mitochondria. Flies with severe reductions in SOD2 expression exhibited accelerated senescence of olfactory behavior as well as precocious neurodegeneration and DNA strand breakage in neurons. Furthermore, life span was progressively shortened and age-dependent mortality was increased in conjunction with reduced SOD2 expression, while initial mortality and developmental viability were unaffected. Interestingly, life span and age-dependent mortality varied exponentially with SOD2 activity, indicating that there might normally be a surplus of this enzyme for protecting animals from premature death. Our data support a model in which disruption of the protective effects of SOD2 on mitochondria manifests as profound changes in behavioral and demographic aging as well as exacerbated age-related pathology in the nervous system.

SOD II loss is associated with aging and accelerated neurologic conditions

Attenuation of myocardial apoptosis by alpha-lipoic acid through suppression of mitochondrial oxidative stress to reduce diabetic cardiomyopathy LI Chun-jun, et al:

Chinese Medical Journal, 2009, Vol. 122 No. 21 : 2580-2586

Cardiac failure is a leading cause of the mortality of diabetic patients. In part this is due to a specific cardiomyopathy, referred to as diabetic cardiomyopathy. Oxidative stress is widely considered to be one of the major factors underlying the pathogenesis of the disease. This study aimed to test whether the antioxidant α-lipoic acid (α-LA) could attenuate mitochondrion-dependent myocardial apoptosis through suppression of mitochondrial oxidative stress to reduce diabetic cardiomyopathy.Results  At 4, 8, and 12 weeks after the onset of diabetes, significant reductions in TUNEL-positive cells, caspase-9,-3 expression, and mitochondrial cytochrome c release were observed in the α-LA group compared to the DM group. In the DM group, the content of MDA in the myocardial mitochondria was significantly increased, and there was a decrease in both the mitochondrial GSH content and the activities of Mn-SOD. They were significantly improved by α-LA treatment. HE staining displayed structural abnormalities in diabetic hearts, while α-LA reversed this structural derangement. The index of cardiac function (±dp/dtmax) in the diabetes group was aggravated progressively from 4 weeks to 12 weeks, but α-LA delayed deterioration of cardiac function (P <0.05). Conclusions  Our findings indicate that the antioxidant α-LA can effectively attenuate mitochondria-dependent cardiac apoptosis and exert a protective role against the development of diabetic cardiomyopathy. The ability of α-LA to suppress mitochondrial oxidative damage is concomitant with an enhancement of Mn-SOD activity and an increase in the GSH content of myocardial mitochondria.

ALA and Protects SOD II Which Protects Mitochondria

FEBS Lett. 2005 Jun 6;579(14):3029-36.

Role of Nrf2 signaling in regulation of antioxidants and phase 2 enzymes in cardiac fibroblasts: protection against reactive oxygen and nitrogen species-induced cell injury.

Zhu H, Itoh K, Yamamoto M, Zweier JL, Li Y.

Room 012C, Davis Heart and Lung Research Institute, The Ohio State University Medical Center, 473 West 12th Avenue, Columbus, OH 43210, United States.

Understanding the molecular pathway(s) of antioxidant gene regulation is of crucial importance for developing antioxidant-inducing agents for the intervention of oxidative cardiac disorders. Accordingly, this study was undertaken to determine the role of Nrf2 signaling in the basal expression as well as the chemical inducibility of endogenous antioxidants and phase 2 enzymes in cardiac fibroblasts. The basal expression of a scope of key cellular antioxidants and phase 2 enzymes was significantly lower in cardiac fibroblasts derived from Nrf2-/- mice than those from wild type control. These include catalase, reduced glutathione (GSH), glutathione reductase (GR), GSH S-transferase (GST), and NAD(P)H:quinone oxidoreductase-1 (NQO1). Incubation of Nrf2+/+ cardiac fibroblasts with 3H-1,2-dithiole-3-thione (D3T) led to a significant induction of superoxide dismutase (SOD), catalase, GSH, GR, glutathione peroxidase (GPx), GST, and NQO1. The inducibility of SOD, catalase, GSH, GR, GST, and NQO1, but not GPx by D3T was completely abolished in Nrf2-/- cells. The Nrf2-/- cardiac fibroblasts were much more sensitive to reactive oxygen and nitrogen species-mediated cytotoxicity. Upregulation of antioxidants and phase 2 enzymes by D3T in Nrf2+/+ cardiac fibroblasts resulted in a dramatically increased resistance to the above species-induced cytotoxicity. In contrast, D3T-treatment of the Nrf2-/- cells only provided a slight cytoprotection. Taken together, this study demonstrates for the first time that Nrf2 is critically involved in the regulation of the basal expression and chemical induction of a number of antioxidants and phase 2 enzymes in cardiac fibroblasts, and is an important factor in controlling cardiac cellular susceptibility to reactive oxygen and nitrogen species-induced cytotoxicity.

Nrf2 increases activity of SOD 2

T3, FREE 2.6 2.6 2.39-6.79 PG/ML

Thyroglobulin Antibody 264H 107 0-25.0 NG/ML

THYROID PEROXIDASE AB. 319 H 195 0-60.0 U/ML

T3-TOTAL 0.69 L 1.03 0.8-2.0 NG/ML

T4, FREE 0.85 1.16 0.58-1.64 NG/DL

THYROID STIM. HORMONE 6.87 H 3.49 0.34-5.60 mIU/L

Reverse T3 152 99 47.7-144 nmol/L

T4 ,TOTAL 6.6 6.6 4.5-12.5 MCG/DL

Thyroid Profile

Peptides. 2004 Jun;25(6):1021-9.Antigenicity and immunogenicity of the C-terminal peptide of

human thyroglobulin.El Hassani RA, Estienne V, Blanchin S, Durand-Gorde JM, Mallet B, De Micco

C, Carayon P, Lalaoui K, Ruf J.

Thyroglobulin (Tg) is cleaved into several peptides during thyroid hormone synthesis, an oxidative process. P40, an iodinated C-terminal peptide from human Tg, has a molecular weight of about 40 kDa and contains two hormonogenic sites. P40 is the smallest peptide that is still recognized by monoclonal antibodies from mice immunized with human Tg directed against its immunodominant region. Since P40 also contains several T-cell epitopes, it is a good candidate for studying the primary events involved in the process of hormone synthesis leading to thyroid autoimmunity. The present results show that P40 is recognized by Tg antibodies from patients with thyroid disorders and induces Tg antibodies in CBA mice. P40 may therefore be involved in the autoimmune process, thus providing a useful tool for diagnostic and therapeutic purposes.

Decreased oxidative stress improves thyroid function

Biochem J. 2001 Dec 15;360(Pt 3):557-62.Hydrogen peroxide-induced production of a 40 kDa immunoreactive thyroglobulin fragment in

human thyroid cells: the onset of thyroid autoimmunity?Duthoit C, Estienne V, Giraud A, Durand-Gorde JM, Rasmussen AK, Feldt-Rasmussen U, Carayon P, Ruf J.

We recently reported that, during in vitro thyroid-hormone synthesis, H(2)O(2) stress cleaved thyroglobulin (Tg) into C-terminal peptides. These peptides were found to contain the immunodominant region of Tg recognized by Tg autoantibodies from patients with an autoimmune thyroid disease. To test the hypothesis that Tg fragmentation is an early upstream initiating event involved in Tg autoimmune response and the consequence of oxidative injuries, we studied the effect of H(2)O(2) stress on human thyroid cells. In culture conditions allowing Tg synthesis and iodine organification by the cells, we found that bolus addition of increasing millimolar doses of H(2)O(2) induced a dose-response appearance of floating cells in the culture medium. These cells apparently resulted from a necrotic process, and they bore iodinated Tg fragments. These fragments were found to be similar to those previously obtained in vitro from purified Tg. In both cases, Tg peptides were recognized by a well-defined monoclonal antibody directed to the immunodominant region of Tg. The smallest immunoreactive Tg peptide had a molecular mass of 40 kDa and entered human thyrocytes more efficiently than the entire Tg. These data suggest that thyrocytes exposed to locally increased H(2)O(2) doses accumulate fragmented Tg for further delivery into surrounding living thyrocytes in the course of an autoimmune response.

Increased glutathione peroxidase helps thyroid and is upregulated by Nrf2

Biol Chem. 2007 Oct;388(10):1053-9.Selenoproteins of the thyroid gland: expression, localization and possible

function of glutathione peroxidase 3.Schmutzler C, Mentrup B, Schomburg L, Hoang-Vu C, Herzog V, Köhrle J.

The thyroid gland has an exceptionally high selenium content, even during selenium deficiency. At least 11 selenoproteins are expressed, which may be involved in the protection of the gland against the high amounts of H2O2 produced during thyroid hormone biosynthesis. As determined here by in situ hybridization and Northern blotting experiments, glutathione peroxidases (GPx) 1 and 4 and selenoprotein P were moderately expressed, occurring selectively in the follicular cells and in leukocytes of germinal follicles of thyroids affected by Hashimoto's thyroiditis. Selenoprotein 15 was only marginally expressed and distributed over all cell types. GPx3 mRNA was exclusively localized to the thyrocytes, showed the highest expression levels and was down-regulated in 5 of 6 thyroid cancer samples as compared to matched normal controls. GPx3 could be extracted from thyroidal colloid by incubation with 0.5% sodium dodecyl sulfate indicating that this enzyme is (i) secreted into the follicular lumen and (ii) loosely attached to the colloidal thyroglobulin. These findings are consistent with a role of selenoproteins in the protection of the thyroid from possible damage by H2O2. Particularly, GPx3 might use excess H2O2 and catalyze the polymerization of thyroglobulin to the highly cross-linked storage form present in the colloid.

Selenium and thyroid function

Adrenal stress testing

11/02/2012

Adrenal stress testing

Psychoneuroendocrinology. 2013 Mar 9. pii: S0306-4530(13)00042-5. doi: 10.1016/j.psyneuen.2013.02.004. [Epub ahead of print]Good stress, bad stress and oxidative stress: Insights from anticipatory cortisol reactivity.Aschbacher K, O'Donovan A, Wolkowitz OM, Dhabhar FS, Su Y, Epel E.SourceDepartment of Psychiatry, University of California San Francisco, San Francisco, CA, USA; The Institute for Integrative Health, Baltimore, MD, USA. Electronic address: kirstin.aschbacher@ucsf.edu.AbstractChronic psychological stress appears to accelerate biological aging, and oxidative damage is an important potential mediator of this process. However, the mechanisms by which psychological stress promotes oxidative damage are poorly understood. This study investigates the theory that cortisol increases in response to an acutely stressful event have the potential to either enhance or undermine psychobiological resilience to oxidativedamage, depending on the body's prior exposure to chronic psychological stress. In order to achieve a range of chronic stress exposure, forty-eight post-menopausal women were recruited in a case-control design that matched women caring for spouses with dementia (a chronic stress model) with similarly aged control women whose spouses were healthy. Participants completed a questionnaire assessing perceived stress over the previous month and provided fasting blood. Three markers of oxidative damage were assessed: 8-iso-prostaglandin F 2α (IsoP), lipid peroxidation, 8-hydroxyguanosine (8-oxoG) and 8-hydroxy-2'-deoxyguanosine (8-OHdG), reflecting oxidative damage to RNA/DNA respectively. Within approximately one week, participants completed a standardized acute laboratory stress task while salivary cortisol responses were measured. The increase from 0 to 30min was defined as "peak" cortisol reactivity, while the increase from 0 to 15min was defined as "anticipatory" cortisol reactivity, representing a cortisol response that began while preparing for the stress task. Women under chronic stress had higher 8-oxoG, oxidative damage to RNA (p<.01). A moderated mediation model was tested, in which it was hypothesized that heightened anticipatory cortisol reactivity would mediate the relationship between perceived stress and elevated oxidative stress damage, but only among women under chronic stress. Consistent with this model, bootstrapped path analysis found significant indirect paths from perceived stress to 8-oxoG and IsoP (but not 8-OHdG) via anticipatory cortisol reactivity, showing the expected relations among chronically stressed participants (p≤.01) Intriguingly, among those with low chronic stress exposure, moderate (compared to low) levels of perceived stress were associated with reduced levels of oxidative damage. Hence, this study supports the emerging model that chronic stress exposure promotes oxidative damage through frequent and sustained activation of the hypothalamic-pituitary-adrenal axis. It also supports the less studied model of 'eustress' - that manageable levels of life stress may enhance psychobiological resilience to oxidative damage.

Adrenals and Oxidative Stress