Dehydroepiandrosterone, Glucose-6-Phosphate Dehydrogenase, and Longevity Arthur G

Ageing Research Reviews 3 (2004) 171–187

Review Dehydroepiandrosterone, glucose-6-phosphate dehydrogenase, and longevity Arthur G. Schwartz a,b,∗, Laura L. Pashko b a Department of Microbiology, Temple University School of Medicine, 3307 North Board Street, Philadelphia, PA 19140, USA b Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA 19140, USA Received 22 May 2003; accepted 28 May 2003

Abstract Dehydroepiandrosterone (DHEA) is an abundantly produced adrenal steroid whose biological role has never been clarified. DHEA is a potent uncompetitive inhibitor of mammalian glucose-6- phosphate dehydrogenase (G6PDH) and as a consequence lowers NADPH levels and reduces NADPH-dependent oxygen-free radical production. Overproduction of oxygen-free radicals, or oxidative stress, upregulates inflammation and cellular proliferation and is believed to play a critical role in the development of cancer, atherosclerosis, and Alzheimer’s disease, as well as the basic aging process. Both in vitro and in vivo experimental studies strongly indicate that DHEA and related steroids inhibit inflammation and associated epithelial hyperplasia, carcinogenesis, and atherosclerosis, at least in part, through the inhibition of G6PDH and oxygen-free radical formation. Recent epidemiological findings in Sardinian males bearing the Mediterranean variant of G6PDH deficiency are consistent with the hypothesis that reduced G6PDH activity has a beneficial effect on age-related disease development and longevity. Clinical trials with DHEA are encumbered by the high oral doses required as well as the conversion of DHEA into active androgens. The use of less androgenic congeners as well as non-oral formulations may facilitate testing of this class of compounds. © 2003 Elsevier Ireland Ltd. All rights reserved.

Keywords: DHEA; G6PDH; Oxygen-free radicals; NADPH; Longevity

1. Introduction

Dehydroepiandrosterone (DHEA) is an abundantly produced adrenal steroid in humans (Parker, 1999). The plasma levels of DHEA and its sulfated ester rise around 6–7 years,

∗ Corresponding author. E-mail address: [email protected] (A.G. Schwartz).

1568-1637/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.arr.2003.05.001 172 A.G. Schwartz, L.L. Pashko / Ageing Research Reviews 3 (2004) 171–187 reach a maximum in the second decade, and thereafter decline to about 15% of the peak level in the ninth decade (Orentreich et al., 1984; Parker, 1999). Roth et al. (2002) recently reported that men in the Baltimore Longitudinal Study on Aging with plasma DHEA-sulfate levels in the upper half of their age group experienced a significantly greater survival than men with DHEA-sulfate levels in lower half of their age group. DHEA is a potent uncompetitive inhibitor of mammalian glucose-6-phosphate dehydrogenase (G6PDH), the first enzyme in the pentose phosphate pathway, a major source of ribose 5-phosphate and extra-mitochondrial NADPH (Raineri and Levy, 1970; Gordon et al., 1995). NADPH is a critical modulator of the cellular redox state and serves as a reductant for several enzymes which generate oxygen-free radicals, including the leukocyte NADPH oxidase (Babior, 1999) and its more widely distributed homolog Nox1 (Suh et al., 1999), nitric oxide synthase (Marletta, 1994), the cytochrome P450 monooxygenases (Sadowski et al., 1985), and the Fenton reaction of iron-mediated catalysis of hy- droxyl radical formation from H2O2 (Imlay and Linn, 1988). Thus, a reduction in the supply of NADPH could have a profound effect on oxygen-free radical production. Ox- idative stress and associated inflammatory processes are believed to play an important role in the pathogenesis of major age-related diseases, such as cancer (Coussens and Werb, 2002), atherosclerosis (Steinberg, 2002), and Alzheimer’s disease (Thomas et al., 1996; Ischiropoulos and Beckman, 2003), as well as the basic aging process (Finkel and Holbrook, 2000). In this perspective article, we review in vitro and in vivo experimental studies which strongly indicate that DHEA and related steroids inhibit inflammation and associated epithelial hyperplasia, carcinogenesis, and atherosclerosis, at least in part, through the inhibition of G6PDH and oxygen-free radical formation. Studies in Sardinian males bearing the Mediterranean variant of G6PDH deficiency, both with cells in vitro and in epidemiologic observations, support the hypothesis that reduced G6PDH activity has a beneficial effect on age-related disease development and longevity. Cultured cells from G6PDH-deficient Sardinians mimic the effect of DHEA treatment: the cells have a lowered intracellular NADPH/NADP+ level and a reduced capacity to activate chemical carcinogens as well as to •− produce superoxide anion (O2 ) when stimulated with the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA). Recent epidemiological studies have found that G6PDH- deficient Sardinian males have a reduced mortality from cerebrovascular and cardiovascular disease and are more likely to achieve centenarian status than their normal counterparts (Fig. 1). The development of DHEA as a potential therapy for the prevention of age-related diseases is hampered by the high oral dosages required as well as by the conversion of DHEA into active androgens. We have developed the synthetic congener, 16␣-fluoro-5-androsten- 17-one (fluasterone), which in preclinical tests lacks the androgenicity of DHEA yet has retained biological efficacy. The effective oral dose of fluasterone, as well as of DHEA, is very high, largely because of first-pass hepatic and/or gastrointestinal metabolism. In animal tests the efficacy of fluasterone is enhanced about 40-fold with the use of various non-oral routes of administration, and a non-oral formulation of fluasterone is currently in clinical trials for the treatment of hypertriglyceridemia and insulin resistance in type 2 diabetic patients. A.G. Schwartz, L.L. Pashko / Ageing Research Reviews 3 (2004) 171–187 173

NADPH — reductant for glucose 6-phosphate NADP+ NADPH various enzymes which generate oxygen-free radicals

6-phosphogluconate

DHEA — uncompetitive inhibitor of G6PDH

ribose 5-phosphate glycolysis and tricarboxylic acid cycle Pentose phosphate pathway

Fig. 1. The inhibition of G6PDH and NADPH production by DHEA and related steroids. DHEA is a potent uncompetitive inhibitor (with respect to NADP+ and glucose-6-phosphate) of mammalian G6PDH and thereby reduces the availability of NADPH and generation of oxygen-free radicals by NADPH-dependent enzymes.

2. Oxidative stress and inﬂammatory processes in cancer and atherosclerosis

2.1. Cancer

Chronic inflammation, due to persistent infection such as hepatitis C, schistosomiasis, or Helicobacter pylori, as well as inflammation of unknown etiology such as inflammatory bowel disease, or inflammation due to non-infective agents, such as asbestosis or reflux esophagitis, are all associated with enhanced risk of developing cancer (Coussens and Werb, 2002). The accumulation of activated leukocytes in inflamed tissue provides an abundant supply of oxygen-free radicals. Two major sources of oxygen-free radicals are the leukocyte NADPH oxidase (Babior, 1999) and inducible nitric oxide synthase, an NADPH-dependent enzyme resembling the cytochrome P450s (Marletta, 1994). Oxygen-free radicals are not only mutagenic (Marnett, 2000), and thus potentially carcinogenic, but also act as intermediate messengers which stimulate cellular proliferation (Sundaresan et al., 1995; Irani et al., 1997) and also further upregulate inflammation (Schreck et al., 1991; Lo et al., 1996). Enhanced cellular proliferation in an environment rich in mutagens is a prime site for neoplastic transformation. Oxygen-free radicals may not only promote cell growth and neoplastic transformation but can also stimulate angiogenesis, thereby enhancing local tissue invasion by tumor cells as well as the development of metastases (Nowicki et al., 1996; Lin et al., 2001). The NADPH oxidase may play an important role in neoplastic development. The leuko- •− cyte version of this multicomponent enzyme catalyzes the production of O2 by the one-electron reduction of oxygen, using NADPH as the electron donor (Babior, 1999). 174 A.G. Schwartz, L.L. Pashko / Ageing Research Reviews 3 (2004) 171–187

The catalytic subunit, gp91phox, is dormant in resting cells but becomes activated by as- •− sembly with cytosolic proteins, leading to the respiratory burst of O2 and other oxidants which kill invading microorganisms. A lower activity oxidase is found in many types of non-phagocytic cells and is believed to provide oxidants for signaling purposes (Babior, 1999; Lambeth et al., 2000). At least five homologs of gp91phox (Nox, non-phagocytic oxidase) have been identified in human tissues and act as subunits of a non-phagocytic oxidase (Lambeth et al., 2000). Non-tumorigenic NIH 3T3 fibroblasts or DU-145 prostate epithelial •− cells transfected with Nox1 showed increased production of O2 and H2O2, enhanced growth rates in vitro, and produced rapidly growing vascularized tumors in athymic mice (Arbiser et al., 2002). Coexpression of catalase along with Nox1 greatly reduced tumori- genicity, indicating that one of the signaling species is H2O2 (Arnold et al., 2001). Nox1 overexpression also markedly up-regulated the expression of vascular endothelial growth factor mRNA as well as matrix metalloproteinase activity, two important markers of the angiogenic switch, which likely contributed to the increased vascularity and high growth rate of the tumors in vivo (Arnold et al., 2001). Interestingly NIH 3T3 cells transfected with human G6PDH cDNA and overexpressing G6PDH activity also exhibit transformed foci and anchorage-independent growth in vitro and produce rapidly growing fibrosarcomas in athymic mice (Kuo et al., 2000). The G6PDH-transfected cells had significantly higher intracellular NADPH levels than non-transfected cells, and treatment of G6PDH-overexpressing cells in vitro with DHEA completely blocked focus formation and significantly reduced anchorage-independent growth (Kuo and Tang, 1998; Kuo et al., 2000).

2.2. Atherosclerosis

Cardiovascular disease, primarily due to atherosclerosis, is the leading cause of death and morbidity in developing countries and is rapidly becoming the major world-wide health problem (Murray and Lopez, 1997). Although earlier views on the pathophysiology of atherosclerosis were dominated by the relationship between plasma lipids and disease development, there is a large body of compelling evidence indicating that inflammation is mechanistically linked to the initiation, progression, and rupture—which precipitates an acute coronary event (Van der Wal et al., 1994)—of atherosclerotic lesions (Libby, 2002). The pathophysiologic significance of elevated plasma cholesterol levels and of inflammation are not mutually exclusive since shortly after administering an atherogenic diet to experimental rabbits light microscopy reveals attachment of blood leukocytes to endothelial cells lining the arterial intima (Poole and Florey, 1958). Oxidized low-density lipoprotein (LDL) is a likely candidate that initiates the inflammatory process, thus producing a link between hypercholesteremia and arterial inflammation (Steinberg, 2002). The attached mononuclear leukocytes migrate into the intima, take on characteristics of tissue macrophages, and incor- porate oxidized LDL to become foam cells. Foam cells secrete oxygen-free radicals as well as various cytokines that further accelerate inflammation. Oxygen-free radical production in the vessel wall mediates, at least in part, oxidation of LDL (Aviram et al., 1996) as well as up-regulation of vascular inflammation (Marumo et al., 1997; Griendling et al., 2000) and growth of vascular smooth muscle cells and fibroblasts (Sundaresan et al., 1995; Griendling et al., 2000; Barry-Lane et al., 2001). A.G. Schwartz, L.L. Pashko / Ageing Research Reviews 3 (2004) 171–187 175

Oxygen-free radicals produced by the NADPH oxidase in vascular smooth muscle cells and fibroblasts may be critically involved in the development of atherosclerosis (Griendling et al., 2000; Barry-Lane et al., 2001). Aortic smooth muscle cells from mice deficient in p47phox, a cytoplasmic subunit of the NADPH oxidase, lack a functional NADPH oxidase •− and show diminished O2 production as well as decreased proliferative response to growth factors compared to wild-type cells (Barry-Lane et al., 2001). When p47phox-deficient mice were bred to ApoE-deficient mice, which have high plasma cholesterol levels and develop spontaneous atherosclerosis, the resultant hybrids developed markedly reduced whole-aortic atherosclerotic lesions when compared to wild-type ApoE-deficient mice (Barry-Lane et al., 2001). Oxygen-free radicals produced by the NADPH oxidase may contribute not only to the pathogenesis of atherosclerosis but also to ischemia-reperfusion injury following arterial oc- clusion leading to a myocardial infarction or stroke (Walder et al., 1997; Ozaki et al., 2000).

3. DHEA Inhibition of Inﬂammation, hyperplasia, and carcinogenesis

3.1. In vitro studies

Most chemical carcinogens require metabolic activation by NADPH-dependent cytochrome P450 monooxygenases to reactive mutagenic and carcinogenic forms (Miller and Miller, 1977). DHEA protects cultured-rat-liver-epithelial-like cells and embryonic fibroblasts against aflatoxin B1- and 7,12-dimethylbenz(a)anthracene (DMBA)-induced cytotoxicity and transformation, whereas related steroids, such as testosterone and etiocholanolone, are significantly less protective (Schwartz and Perantoni, 1975). DHEA treatment also suppresses the rate of metabolism of [3H] DMBA to water-soluble products by cultured cells, and epiandrosterone, a more potent G6PDH inhibitor than DHEA (Raineri and Levy, 1970), is more active in reducing [3H] DMBA metabolism. We hypothesized that DHEA protects cultured cells against carcinogen-induced cytotoxicity and transformation by reducing carcinogen activation as a result of inhibiting NADPH production (Schwartz and Perantoni, 1975). Lee et al. (1993) reached a similar conclusion in their studies with paraquat-induced cytotoxicity in cultured-rat-tracheal-epithelial cells. Paraquat, a herbicide and pneumotoxi- •− cant, undergoes redox cycling with NADPH-dependent diaphorases to generate O2 from O2 (Misra and Gorsky, 1981; Day et al., 1999). Treatment of tracheal cells with DHEA or epiandrosterone significantly decreased intracellular NADPH levels and protected the cells against paraquat-induced toxicity (Lee et al., 1993). •− Neutrophils, when treated with the tumor-promoter TPA, rapidly produce O2 through the action of the leukocyte NADPH oxidase (Babior, 1999). Treatment of human neu- •− trophils with DHEA or 16␣-bromoepiandrosterone inhibits the rate of TPA-induced O2 production as well as G6PDH activity in cell lysates (Whitcomb and Schwartz, 1985). 16␣-Bromoepiandrosterone is a more powerful inhibitor of both G6PDH activity (Raineri •− •− and Levy, 1970) and O2 formation, suggesting that the steroids reduce O2 generation by limiting NADPH production. As discussed earlier the NADPH oxidase may be causally linked to the pathogenesis of atherosclerosis and cancer (Griendling et al., 2000; Barry-Lane et al., 2001; Arbiser et al., 2002). 176 A.G. Schwartz, L.L. Pashko / Ageing Research Reviews 3 (2004) 171–187

DHEA protection against carcinogen- and paraquat-induced toxicity, as well as the re- •− duction in O2 production in TPA-treated neutrophils, very likely results from a reduction in NADPH availability. However, in other experimental systems DHEA may inhibit the growth and differentiation of cultured cells by limiting the synthesis of ribose 5-phosphate. Addition of a mixture of the four deoxyribonucleosides (DRN) to the culture medium completely reversed DHEA-induced growth inhibition of HeLa TCRC-2 cells (Dworkin et al., 1988), and treatment with a mixture of the four ribonucleosides (RN) overcame the block- ing action of 16␣-bromoepiandrosterone on the differentiation of 3T3-L1 fibroblasts into adipocytes (Gordon et al., 1987). Shantz et al. (1989) provided direct evidence that treatment of 3T3-L1 fibroblasts with DHEA and various congeners inhibited the pentose phosphate pathway leading to a block in the differentiation into adipocytes. They found that treatment of 3T3-L1 cells with the potent G6PDH inhibitor 16␣-bromoepiandrosterone reduced the intracellular levels of 6-phospho-␦-gluconolactone and other intermediates of the pentose phosphate pathway. Introduction of 6-phospho-␦-gluconolactone incorporated into liposomes into the cells raised the intracellular levels of 6-phospho-␦-gluconolactone and other pentose phosphate pathway sugar phosphates and partially restored the steroid-induced block in growth and differentiation.However, other investigators found that DHEA treatment of various cell lines suppressed cellular proliferation induced by growth factors, such as epidermal growth factor, platelet-derived growth factor, and serum, and that this growth inhibition was not overcome by treatment with a mixture of the four DRN or four RN (Tian et al., 1998). They also found that DHEA treatment of cells reduced the NADPH/NADP+ ratio about two-fold and concluded that NADPH was the likely pentose phosphate pathway product critical for growth stimulation (Tian et al., 1998). As described below, the observation that treatment of mice with a NADPH–liposome mixture reverses DHEA-analog suppression of TPA-induced epidermal inflammation and hyperplasia is consistent with this hypothesis (Schwartz and Pashko, 2001).

3.2. In vivo studies

DHEA treatment of mice and rats inhibits the development of spontaneous, chemically- induced, and radiation-induced tumors in numerous organs. A partial listing includes breast cancer in C3H mice (Schwartz, 1979), methylnitrosourea (MNU)-induced (Ratko et al., 1991), DMBA-induced (Li et al., 1993), and radiation-induced (Inano et al., 1995) mammary cancer in rats, diethylnitrosamine-induced persistent liver nodules in rats subjected to the resistant-hepatocyte protocol (Simile et al., 1995), DMBA- and urethan-induced lung tumors in mice (Schwartz and Tannen, 1981), 1,2-dimethylhydrazine-induced colon tumors in mice (Nyce et al., 1984), MNU-induced prostate cancer in rats (Rao et al., 1999), spontaneous lymphomas and hepatomas in p53-deficient mice (Perkins et al., 1997), and spontaneous testicular Leydig cell tumors in aging rats (Rao et al., 1992). Topical application of DHEA to the backs of mice inhibits the development of DMBA-initiated and TPA-promoted skin papillomas at both the initiation and promotion stages and also suppresses the formation of skin papillomas and carcinomas produced by multiple applications of DMBA (Pashko et al., 1984; Pashko et al., 1985). Both the suppression of tumor initiation and tumor promotion by the DHEA steroids is very likely due to inhibition of G6PDH. Topical application of DHEA or 3␤-methyl-5- A.G. Schwartz, L.L. Pashko / Ageing Research Reviews 3 (2004) 171–187 177 androsten-17-one, a more potent G6PDH inhibitor, to the backs of mice inhibits the binding of [3H] DMBA to epidermal DNA, very probably by reducing carcinogen activation by NADPH-dependent cytochrome P450 monooxygenases (Pashko et al., 1984). This mechanism may account for the inhibition in papilloma and carcinoma development when steroid is applied shortly before carcinogen (Pashko et al., 1985). DHEA and two synthetic congeners, 3␤-methyl-5-androsten-17-one and fluasterone, also inhibit TPA-promoted epidermal papilloma formation when applied topically 1 h before each application of TPA (Pashko et al., 1984; Pashko et al., 1991). TPA, a protein kinase C agonist, induces both acute inflammation and epidermal hyperplasia in mouse skin (Lee et al., 1994). Both of these responses are suppressed by treatment with fluasterone (Schwartz and Pashko, 2001), a potent G6PDH inhibitor with a Ki of 0.5 ␮M versus 17 ␮M for DHEA (Schwartz et al., 1988). As discussed earlier, DHEA-induced inhibition of growth and differentiation of cultured cells is overcome by the presence of a mixture of the four DRN or four RN in the culture medium (Gordon et al., 1987; Dworkin et al., 1988). Garcea et al. (1988) found that in- traperitoneal injection of a mixture of the four DRN or four RN in rats reversed the growth inhibitory effect of DHEA treatment on preneoplastic liver focus formation in vivo. We observed a similar effect of DRN treatment on the promotion of skin papillomas in mice. Topical application of fluasterone to the backs of mice suppressed TPA-induced epidermal hyperplasia and inhibited papilloma development. Treatment with a mixture of four DRN in the drinking water completely reversed the inhibition in TPA-induced hyperplasia and tumor promotion produced by fluasterone (Pashko et al., 1991). These data strongly suggest that inhibition of G6PDH by DHEA steroids is a critical mechanism by which the steroids inhibit tumor-promoter-induced hyperplasia and development of tumors. Although the above experiments suggest that DHEA steroids retard tumor promotion through the inhibition of nucleic acid synthesis as a result of reducing the supply of ribose 5-phosphate, this mechanism may only partly account for the inhibition in tumor development. We have found that dosages of fluasterone in rats as high as 12 times the dose needed to inhibit TPA-stimulated epidermal hyperplasia produce no apparent toxicity and do not suppress mitogenesis in rapidly proliferating tissues (Pallman and Ackerman, 1996), suggesting that the anti-proliferative effect of fluasterone may be directly linked to its anti-inflammatory action and may be more a result of inhibition in oxygen-free radical production rather than a reduction in nucleotide synthesis. As mentioned previously, there is increasing evidence that oxygen-free radicals act as intermediate messengers which stimulate mitogenesis and upregulate inflammation.

4. Suppression of inﬂammation and hyperplasia and reversal by NADPH–liposomes

Topical application of a single dose of TPA to mouse skin induces intense inflammation and epidermal hyperplasia (Lee et al., 1994). Treatment with topical fluasterone or the anti-inflammatory glucocorticoid corticosterone prior to TPA application suppresses the inflammatory and hyperplastic response (Schwartz and Pashko, 2001). Since NADPH, a negatively charged dinucleotide, does not penetrate cells we sought a procedure for enhancing cellular uptake of NADPH to determine its effect on steroid-induced suppression of inflammation and hyperplasia. We used a commercial preparation of cationic liposomes 178 A.G. Schwartz, L.L. Pashko / Ageing Research Reviews 3 (2004) 171–187 which interact spontaneously with polynucleotides and is a highly effective transfection agent as a means of facilitating NADPH entrance into cells. We reasoned cationic liposomes would also interact spontaneously with the dinucleotide NADPH and act as a deliv- ery vehicle for the normally impenetrable dinucleotide. We found that intradermal injection of NADPH–liposomes completely reversed the anti-inflammatory and anti-hyperplastic effects of fluasterone in TPA-treated mouse skin (Schwartz and Pashko, 2001). Similar in- jections of an NADPH solution without liposomes or liposomes without NADPH had no apparent effect on fluasterone activity. Whereas NADPH–liposome treatment reversed the suppressive effect of fluasterone on TPA-induced inflammation and hyperplasia, similar treatment had no apparent effect on the suppressive action of corticosterone. These experiments strongly suggest that fluasterone exerts its anti-inflammatory and anti-hyperplastic action through the inhibition of G6PDH and consequent reduction in NADPH levels, whereas corticosterone, a classical glucocorticoid and anti-inflammatory steroid, does not inhibit G6PDH (Marks and Banks, 1960) and exerts its anti-inflammatory action through a different mechanism (DeBosscher et al., 2000). We also observed that DRN treatment reversed the anti-inflammatory as well as the anti-hyperplastic effect of fluasterone (Schwartz and Pashko, 1993). A possible interpretation of this latter finding is that DRN treatment not only replaces depleted nucleotide pools but also changes the redox state of the cells since NADPH is no longer needed for ribonucleotide and deoxyribonucleotide synthesis.

5. Inhibition of atherosclerosis

DHEA treatment inhibits atherosclerosis development in rabbits. In a model of severe atherosclerosis produced by balloon-catheter-induced aortic endothelial injury followed by feeding a 2% cholesterol diet for 12 weeks, DHEA treatment reduced plaque size by almost 50% (Gordon et al., 1988). These beneﬁcial effects of DHEA were not attributable to differences in body weight, food intake, or plasma cholesterol levels. DHEA treatment also reduced aortic-fatty-streak development in cholesterol-fed rabbits without vascular injury, also without any apparent effect on plasma cholesterol levels (Arad et al., 1989). Lastly, in a hypercholesterolemic model of heterotropic cardiac transplantation which mimics the accelerated atherosclerosis seen in heart transplantation patients, DHEA treatment reduced the number of diseased arterial branches, the severity of luminal stenoses, and the intimal content of lipid-laden foam cells (Eich et al., 1993).

6. G6PDH deﬁciency

The gene encoding for G6PDH is located in the telomeric region of the long arm of the X-chromosome and is flanked on either side by factor VIII coagulant protein and the red/green color pigment genes (Vulliamy et al., 1992). Over 34 polymorphic G6PDH variants resulting in decreased erythrocyte enzymatic activity have been described (Luzzatto et al., 2001). These polymorphic variants are found in populations residing in malarially- endemic tropical and subtropical regions (Beutler, 1994), and the reduced erythrocyte G6PDH activity is believed to confer resistance to Plasmodium falciparum (Cappadoro A.G. Schwartz, L.L. Pashko / Ageing Research Reviews 3 (2004) 171–187 179 et al., 1998). The Mediterranean variant (Gd-Mediterranean) is attributable to a C → T transition in nucleotide 563 causing a Ser → Phe amino acid change in position 188 of the protein and resulting in decreased enzyme stability and reduced catalytic efficiency (Vulliamy et al., 1988; DeVita et al., 1989). The Gd-Mediterranean allele occurs in about 12% of the general male Sardinian population and is associated with an erythrocyte enzyme activity that is undetectable by routine methods (Siniscalco et al., 1961; Cocco et al., 1998). Non-erythrocyte cells, such as neutrophils (Pascale et al., 1987), cultured fibroblasts (Feo et al., 1984), cultured lymphocytes (Feo et al., 1987), and lung tissue (Dessi et al., 1988) also have very low levels of enzyme activity, on the order of 8–15% of normal. Feo et al. (1984, 1987) found that cultured fibroblasts and lymphocytes from G6PDH- deficient male Sardinians were less sensitive to the toxic and transforming effects of the carcinogen benzo(a)pyrene (BP) than normal cells. The deficient cells also demonstrated a marked reduction in the NADPH/NADP+ ratio as well as a reduced capacity to produce BP-water-soluble, DNA-binding, and mutagenic metabolites (Feo et al., 1984; Feo et al., 1987). DHEA treatment of normal fibroblasts and lymphocytes in vitro mimicked the effect of G6PDH deficiency. In addition neutrophils from G6PDH-deficient individuals, •− when stimulated with TPA, produced 60% less O2 than stimulated normal cells, again mimicking the effect of DHEA treatment (Whitcomb and Schwartz, 1985; Pascale et al., 1987). Individuals with G6PDH deficiency may develop hemolytic anemia when exposed to certain drugs or fava beans (Beutler, 1994); otherwise these individuals appear healthy and many reach adulthood without an awareness of their deficient phenotype (Cocco et al., 1998). In 1981 the Health Department of the Regional Administration of Sardinia launched a program of testing of the general population for G6PDH deficiency to reduce the like- lihood of hemolytic crises following ingestion of fava beans. Approximately 2% of the resident population participated in the screening, and among these a cohort of 1756 men expressed a complete enzymatic deficiency in erythrocytes. Eleven years later, vital status was determined for 97% of the cohort, who experienced 121 deaths (Cocco et al., 1998). Death certificates were available for 97% of the deceased, and the primary causes of death and total mortality were compared to that expected for the general male Sardinian population from reference rates made available by the Italian National Institute of Health. The G6PDH-deficient cohort experienced a significantly reduced overall mortality (76% of expected), largely due to a four-fold reduction in mortality from cerebrovascular disease and ischemic heart disease. However, since the study cohort was not a random sample of the general male Sardinian population, selection bias could not be ruled out. A much greater proportion of women than men reach the age of 100 and beyond (centenarians). In continental Italy, the female:male ratio is 4:1 (Franceschi et al., 2000) and in a population of 300 French centenarians the ratio was 8:1 (Schachter et al., 1994). Remark- ably in Sardinia the ratio is 2:1 (Deiana et al., 1999), a finding that was confirmed by an ad-hoc European Committee (Passarino et al., 2001). This unusual proportion was not due to a decreased prevalence of female centenarians in Sardinia but to a very high prevalence of male centenarians. The prevalence of male centenarians in Sardinia is more than three times that reported in continental Italy (Franceschi et al., 2000). As part of the Sardinian Centenarian Study, Deiana et al. (2000) collected blood samples from 123 Sardinian centenarians (39 men, 84 women) as well as from 150 unrelated exactly 180 A.G. Schwartz, L.L. Pashko / Ageing Research Reviews 3 (2004) 171–187

60-year-old individuals from the same geographic area. The diagnosis of G6PDH deficiency was assessed, and the G6PDH genotype was determined on DNA isolated from peripheral leukocytes, amplified by PCR, and analyzed by Mbo II digestion (G6PDH 563 C/T). They found that 25.5% of centenarian males had the 563 C/T variant compared to 11.5% of the 60-year-old controls. Considering the high prevalence of G6PDH deficiency in Sardinia, these findings may help explain the unusual longevity experienced by Sardinian males.

7. Other mechanisms—anti-glucocorticoid action

In addition to the anti-inﬂammatory, cancer preventive, and anti-atherosclerotic actions the DHEA steroids produce biological effects in laboratory animals which are apparently not mediated by G6PDH inhibition and which share the common property of antagonizing certain biological responses produced by the glucocorticoids (Kalimi et al., 1994). Since these glucocorticoid-induced physiologic effects are deleterious, and since in humans cortisol levels rise with age whereas DHEA levels decrease, leading to a marked decline in the DHEA:cortisol ratio, this has led to the hypothesis that DHEA could be used to treat speciﬁc age-related conditions that might be caused, at least in part, by excess cortisol action (Lauglin and Barrrett-Connor, 2000). Excess glucocorticoid exposure suppresses immune function (Crabtree et al., 1979), causes hippocampal atrophy with consequent memory dysfunction (Sapolsky, 1985; Lupien et al., 1998), and induces an obese, diabetic, hyperlipidemic state known as the metabolic syndrome (Pecke and Chrousos, 1995; Masuzaki et al., 2001). In laboratory animals DHEA treatment protects against dexamethasone-induced thymic involution (Blauer et al., 1991), inhibits the neurotoxic actions of glutamate agonists in the rat hippocampus (Kimonides et al., 1998) as well as stimulates hippocampal neurogenesis and protects against corticosterone-induced suppression of neurogenesis (Karisma and Herbert, 2002), and reduces insulin resistance in diabetic mice (Coleman et al., 1982). The mechanism of this anti-glucocorticoid action is not known and presumably is mediated by interaction with a receptor(s) other than G6PDH.

8. Potential therapeutic use of DHEA and analogs

The preclinical findings with DHEA and analogs demonstrating inhibition of carcinogenesis and atherosclerosis as well as the amelioration of various age-related deleterious physiologic changes due, at least in part, to excess cortisol exposure, such as immunosenescence, hippocampal atrophy, and insulin resistance, suggest potentially important therapeutic application for the treatment and prevention of age-related diseases. The oral doses of DHEA given to laboratory animals to prevent cancer, inhibit atherosclerosis, and ameliorate insulin resistance are very high, on the order of 200–400 mg/kg daily (Svec and Porter, 1998). An estimation of the human oral dose would be in the range of 1000–2000 mg daily (Freireich et al., 1966). In a double-blind-placebo-controlled crossover study in six postmenopausal women for 4 weeks, DHEA oral treatment at 1600 mg daily significantly elevated plasma testosterone and dihydrotestosterone levels 9-fold and 20-fold, respectively (Mortola and Yen,1990). Following DHEA treatment plasma HDL levels decreased by 20–30%, and peak A.G. Schwartz, L.L. Pashko / Ageing Research Reviews 3 (2004) 171–187 181 insulin levels during a 3 h-glucose tolerance test were significantly higher and were associated with a 50% increase in the integrated insulin response. Thus, significant metabolism of DHEA into active androgens may greatly limit the use of high dose oral DHEA therapy. In addition to sex-hormonal side-effects, DHEA treatment stimulates liver peroxisome formation and produces hepatomegaly in mice and rats (Frenkel et al., 1990), and long-term administration of the steroid to rats induces a high incidence of hepatocellular carcinoma (Rao et al., 1992). We have developed the synthetic steroid, fluasterone, which in mice and rats does not demonstrate the androgenic, estrogenic, or peroxisomal-proliferating effects (Schwartz et al., 1988) of DHEA yet has retained the antiinflammatory, anti-hyperplastic, cancer preventive, anti-obesity, and anti-diabetic activity of the native steroid (Schwartz et al., 1988, 1989; Pashko et al., 1991; Ratko et al., 1991; Pashko and Schwartz, 1993; Perkins et al., 1997; Schwartz and Pashko, 2001). Fluasterone, when administered at an oral dose + of 200 mg/kg daily to BKS.Cg-m /+ Leprdb mice, which produce a truncated leptin receptor with impaired signaling capacity and are highly insulin resistant (Chen et al., 1996), markedly lowers fasting plasma glucose and triglyceride levels (Pashko and Schwartz, 1993; Schwartz and Pashko, unpublished observation). In early clinical trials in patients with elevated triglycerides (300–900 mg/dl) treatment with 1200 mg fluasterone daily significantly lowers fasting plasma triglycerides, a therapeutic effect similar to that produced in diabetic mice (Kane et al., unpublished observation). Fluasterone, like DHEA, requires high oral doses in animals to produce efficacy. The lowest effective oral dose of fluasterone which abolishes TPA-stimulated epidermal hyperplasia in mouse skin is 200 mg/kg, whereas when administered by various non-oral routes the lowest effective dose is 2.5–5 mg/kg (Schwartz and Pashko, unpublished observation). + The same dose relationship was observed in BKS.Cg-m /+ Leprdb mice for efficacyinlow- ering fasting plasma glucose and triglyceride levels. Orally-administered 14C-fluasterone undergoes extensive first-pass hepatic and/or gastrointestinal metabolism, thus necessitating high oral doses to achieve efficacy (Schwartz and Pashko, unpublished observation). Cur- rently a non-oral formulation of fluasterone is being tested in clinical trials for the treatment of hypertriglyceridemia and hyperglycemia in patients with type 2 diabetes.

9. Conclusion

The mechanistic studies with DHEA steroids suggesting the importance of G6PDH inhibition in reducing inflammation, hyperplasia, and carcinogenesis, the impaired capacity of cultured cells from G6PDH-deficient individuals to activate chemical carcinogens and to •− produce O2 , and the reduced mortality among G6PDH-deficient Sardinians as well as the greater prevalence of G6PDH deficiency among centenarians all concur and suggest that G6PDH inhibition may retard the rate of development of age-related diseases. Although it is likely that oxidative stress and inflammation are causally linked to the biology of aging (Finkel and Holbrook, 2000), the evidence for this is perhaps less compelling than it is for specific age-related diseases, such as cancer and atherosclerosis. Olshansky et al. (2002) have estimated that eliminating all age-related causes of death in the elderly as currently stated on death certificates would only increase life expectancy by 15 years at most. Whether 182 A.G. Schwartz, L.L. Pashko / Ageing Research Reviews 3 (2004) 171–187 the enhanced longevity of G6PDH-deficient Sardinians is due to a delay in the development of age-related diseases or to a delay in the process of aging per se may be ascertainable in future epidemiological studies. Ultimately the therapeutic use of specific DHEA analogs may be of value in retarding the rate of development of various age-related diseases.

Acknowledgements

Work in this laboratory has been supported by grants from the NIH, the American Institute for Cancer Research, and Aeson Therapeutics, Inc.

References

Arad, Y., Badimon, J.J., Badimon, L., Hembree, W.C., Ginsberg, H.N., 1989. Dehydroepiandrosterone feeding prevents aortic fatty streak formation and cholesterol accumulation in cholesterol-fed rabbit. Arteriosclerosis 9, 159–166. Arbiser, J.L., Petros, J., Klafter, R., Govindajaran, B., McLaughlin, E.R., Brown, L.F., Cohen, C., Moses, M., Kilroy, S., Arnold, R.S., Lambeth, J.D., 2002. Reactive oxygen generated by Nox1 triggers the angiogenic switch. Proc. Natl. Acad. Sci. U.S.A. 99, 715–720. Arnold, R.S., Shi, J., Murad, E., Whalen, A.M., Sun, C.Q., Polavarapu, R., Parthasarathy, S., Petros, J.A., Lambeth, J.D., 2001. Hydrogen peroxide mediates the cell growth and transformation caused by the mitogenic oxidase Nox1. Proc. Natl. Acad. Sci. U.S.A. 98, 5550–5555. Aviram, M., Rosenblat, M., Etzioni, A., Levy, R., 1996. Activation of NADPH oxidase required for macrophage-mediated oxidation of low-density lipoprotein. Metabolism 45, 1069–1079. Babior, B.M., 1999. NADPH-oxidase: an update. Blood 93, 1464–1476. Barry-Lane, P.A., Patterson, C., van der Merwe, M., 2001. P47 phox is required for atherosclerotic lesions progression in ApoE−/− mice. J. Clin. Invest. 108, 1513–1522. Beutler, E., 1994. G6PD deficiency. Blood 84, 3613–3636. Blauer, K.L., Poth, M., Rogers, W.M., Benton, E.W., 1991. Dehydroepiandrosterone antagonizes the suppressive effects of dexamethasone on lymphocyte proliferation. Endocrinology 129, 3174–3179. Cappadoro, M., Giribaldi, G., O’Brien, E., Turrini, F., Mannu, F., Ulliers, D., Simula, G., Luzzatto, L., Arese, P., 1998. Early phagocytosis of glucose-6-phosphate dehydrogenase (G6PD)-deficient erythrocytes parasitized by Plasmodium falciparum may explain malaria protection in G6PD deficiency. Blood 92, 2527–2534. Chen, H., Charlat, O., Tartaglia, L.A., Woolf, E.A., Weng, X., Ellis, S.J., Lakey, N.D., Culpepper, J., Moore, K.J., Breitbart, R.E., Duyk, G.M., Tepper, R.I., Morgenstern, J.P., 1996. Evidence that the diabetes gene encodes the leptin receptor: identification of a mutation in the leptin receptor in db/db mice. Cell 84, 491–495. Cocco, P., Todde, P., Fornera, S., Manca, M.B., Manca, P., Sais, A.R., 1998. Mortality in a cohort of men expressing the glucose-6-phosphate dehydrogenase deficiency. Blood 91, 706–709. Coleman, D.L., Leiter, E.H., Schwizer, R.W., 1982. Therapeutic effects of dehydroepiandrosterone (DHEA) on diabetic mice. Diabetes 31, 830–833. Coussens, L.M., Werb, Z., 2002. Inflammation and cancer. Nature 420, 860–867. Crabtree, G.R., Gillis, S., Smith, K.A., Munck, A., 1979. Glucocorticoids and immune responses. Arthritis Rheum. 22, 1246–1256. Day, B.J., Patel, M., Calavetta, L., Stamler, J.S., 1999. A mechanism of paraquat toxicity involving nitric oxide synthase. Proc. Natl. Acad. Sci. U.S.A. 96, 12760–12765. DeBosscher, K., Berghe, W.V., Vermeulen, L., Plaisance, S., Boone, E., Haegeman, G., 2000. Glucocorticoids repress NF-␬B-driven genes by disturbing the interaction of p65 with the basal transcription machinery, irrespective of coactivator levels in the cell. Proc. Natl. Acad. Sci. U.S.A. 97, 3919–3924. Deiana, L., Pes, G.M., Carru, C., Errigo, A., Simula, M.P., Perino, M.G., Mura, A., Franceschi, C., Baggio, G., 2000. ␤-Thalassemia and G6PD deficiency are associated with increased longevity in Sardinia. Clin. Chem. 46, A210. A.G. Schwartz, L.L. Pashko / Ageing Research Reviews 3 (2004) 171–187 183

Deiana, L., Ferrucci, L., Pes, G.M., Carru, C., Delitala, G., Ganau, A., Mariotti, S., Nieddu, A., Pettinato, S., Putzu, P., Franceschi, C., Baggio, G., 1999. AKEntAnnos. The Sardinia Study of extreme longevity. Aging (Milano) 11, 142–149. Dessi, S., Batetta, B., Cherchi, R., Onnis, R., Pisano, M., Pani, P., 1988. Hexose monophosphate shunt enzymes in lung tumors from normal and glucose-6-phosphate-dehydrogenase-deficient subjects. Oncology 45, 287–291. DeVita, G., Alcalay, M., Sampietro, M., Cappelini, M.D., Fiorelli, G., Toniolo, D., 1989. Two point mutations are responsible for G6PD polymoprhism in Sardinia. Am. J. Genet. 44, 233–240. Dworkin, C.R., Gorman, S.D., Pashko, L.L., Cristofalo, V.J., Schwartz, A.G., 1988. Inhibition of growth of HeLa and WI-38 cells by dehydroepiandrosterone and its reversal by ribo- and deoxyribonucleosides. Life Sci. 38, 1451–1457. Eich, D.M., Nestler, J.E., Johnson, D.E., Dworkin, G.H., Ko, D., Wechsler, A.S., Hess, M.L., 1993. Inhibition of accelerated atherosclerosis with dehydroepiandrosterone in the heterotropic rabbit model of cardiac transplantation. Circulation 87, 261–269. Feo, F., Pirisi, L., Pascale, R., Daino, L., Frassetto, S., Garcea, R., Gaspa, L., 1984. Modulatory effect of glucose-6-phosphate dehydrogenase deficiency on benzo(a)pyrene toxicity and transforming activity for in vitro-cultured human skin fibroblasts. Cancer Res. 44, 3419–3425. Feo, F., Ruggiu, M.E., Lenzerini, L., Garcea, R., Daino, L., Frassetto, S., Addis, V., Gaspa, L., Pascale, R., 1987. Benzo(a)pyrene metabolism by lymphocytes from normal individuals and individuals carrying the Mediterranean variant of glucose-6-phosphate dehydrogenase. Int. J. Cancer 39, 560–564. Finkel, T., Holbrook, N.J., 2000. Oxidants, oxidative stress, and the biology of ageing. Nature 408, 239–247. Franceschi, C., Motta, L., Valensin, S., Rapisarda, R., Franzone, A., Berardelli, M., Motta, M., Monti, D., Bonafe, M., Ferrucci, L., Deiana, L., Pes, G.M., Carru, C., Desole, M.S., Barbi, C., Sartoni, G., Gemelli, C., Lescai, F., Olivieri, F., Marchegiani, F., Cardelli, M., Cavallone, L., Gueresi, P., Cossarizza, A., Troiano, L., Pini, G., Sansoni, P., Passeri, G., Lisa, R., Spazzafumo, L., Amadio, L., Giunta, S., Stecconi, R., Morresi, R., Viticchi, C., Mattace, R., De Benedictis, G., Baggio, G., 2000. Do men and women follow different trajectories to reach extreme longevity? Italian Multicenter Study on Centenarians (IMUSCE). Aging (Milano) 12, 77–84. Freireich, E.J., Gehan, E.A., Rall, D.P., Schmidt, L.H., Skipper, H.E., 1966. Quantitative comparison of toxicity of anticancer agents in mouse, rat, hamster, dog, monkey and man. Cancer Chemother. Rep. 50, 219–244. Frenkel, R.A., Slaughter, C.A., Orth, K., Moomaus, C.R., Hicks, S.H., Synder, J.M., Bennett, M., Prough, R.A., Putnam, R.S., Milewich, L., 1990. Peroxisome proliferation and induction of peroxisomal enzymes in mouse and rat liver by dehydroepiandrosterone feeding. J. Steroid Biochem. 35, 333–342. Garcea, R., Daino, L., Frassetto, S., Cozzolino, P., Ruggiu, M., Yannini, M.G., Pascale, R., Lenzerini, L., Simile, M.M., Puddu, M., Feo, F., 1988. Reversal by ribo- and deoxyribonucleosides of dehydroepiandrosterone-induced inhibition of enzyme altered foci in the liver of rats subjected to the initiation-selection process of experimental carcinogenesis. Carcinogenesis 9, 931–938. Gordon, G.B., Shantz, L.M., Talalay, P., 1987. Modulation of growth, differentiation and carcinogenesis by dehydroepiandrosterone. Adv. Enzyme Regul. 26, 355–382. Gordon, G.B., Bush, D.E., Weisman, H.F., 1988. Reduction of atherosclerosis by administration of dehydroepiandrosterone. J. Clin. Invest. 82, 712–720. Gordon, G.B., MacKow, M.C., Levy, H.R., 1995. On the mechanism of interaction of steroids with human glucose-6-phosphate dehydrogenase. Arch. Biochem. Biophys. 318, 25–29. Griendling, K.K., Sorescu, D., Ushio-Fukai, M., 2000. NADPH oxidase: role in cardiovascular biology and disease. Circulation Res. 86, 494–501. Imlay, J.A., Linn, S., 1988. DNA damage and oxygen radical toxicity. Science 240, 1302–1309. Inano, H., Ishii-Ohba, H., Suzuki, K., Yamanouchi, H., Onoda, M., Wakabayashi, K., 1995. Chemoprevention by dietary dehydroepiandrosterone against promotion/progression phase of radiation-induced mammary tumorigenesis in rats. J. Steroid Biochem. Mol. Biol. 54, 47–53. Irani, K., Xia, Y., Zweier, J.L., Sollott, S.J., Der, C.J., Fearon, E.R., Sundaresan, M., Finkel, T., Goldschmidt-Clermont, P.J., 1997. Mitogenic signaling mediated by oxidants in ras-transformed fibroblasts. Science 275, 1649–1652. Ischiropoulos, H., Beckman, J.S., 2003. Oxidative stress and nitration in neurodegeneration: Cause, effect, or association? J. Clin. Invest. 111, 163–169. Kalimi, M., Shafagoj, Y., Loria, R., Padgett, D., Regelson, W., 1994. Anti-glucocorticoid effects of dehydroepiandrosterone (DHEA). Mol. Cell. Biochem. 131, 99–104. 184 A.G. Schwartz, L.L. Pashko / Ageing Research Reviews 3 (2004) 171–187

Karisma, K.K., Herbert, J., 2002. Dehydroepiandrosterone (DHEA) stimulates neurogenesis in the hippocampus of the rat, promotes survival of newly formed neurons, and prevents corticosterone-induced suppression. Eur. J. Neurosci. 16, 445–453. Kimonides, V.G., Khatibi, N.H., Svendsen, C.N., Sofroniew, M.V., Herbert, J., 1998. Dehydroepiandrosterone (DHEA) and DHEA-sulfate (DHEAS) protect hippocampal neurons against excitatory amino acid-induced neurotoxicity. Proc. Natl. Acad. Sci. U.S.A. 95, 1852–1857. Kuo, W., Tang, T.K., 1998. Effects of G6PD overexpression in NIH 3T3 cells treated with tert-butyl hydroperoxide or paraquat. Free Radic. Biol. Med. 24, 1130–1138. Kuo, W., Lin, J., Tang, T.K., 2000. Human glucose-6-phosphate dehydrogenase (G6PD) gene transforms NIH 3T3 cells and induces tumors in nude mice. Int. J. Cancer 85, 857–864. Lambeth, J.D., Cheng, G., Arnold, R.S., Edens, W.A., 2000. Novel homologs of gp91phox. Trends Biochem. Sci. 25, 459–461. Lauglin, G.A., Barrrett-Connor, E., 2000. Sexual dimorphism in the influence of advanced aging on adrenal hormone levels: The Rancho Bernardo Study. J. Clin. Endocrinol. Metab. 85, 3561–3568. Lee, T.-C., Lai, G.-J., Kao, S.-L., Ho, I.-C., Wu, C.-W., 1993. Protection of a rat tracheal epithelial cell line from paraquat toxicity by inhibition of glucose-6-phosphate dehydrogenase. Biochem. Pharmacol. 45, 1143–1147. Lee, W.Y., Locknisar, M.F., Fischer, S.M., 1994. Interleukin-1␣ mediates phorbol-ester-induced inflammation and epidermal hyperplasia. FASEB J. 8, 1081–1087. Li, S., Xun, Y., Bélanger, A., Labrie, F., 1993. Prevention by dehydroepiandrosterone of the development of mammary carcinoma induced by 7,12-dimethylbenz(a)anthracene (DMBA) in the rat. Breast Cancer Res. Treat. 29, 203–217. Libby, P., 2002. Inflammation in atherosclerosis. Nature 420, 868–874. Lin, E.Y., Nguyen, A.V., Russell, R.G., Pollard, J.W., 2001. Colony-stimulating factor I promotes progression of mammary tumors to malignancy. J. Exp. Med. 196, 727–740. Lo, Y.Y.C., Wong, J.M.S., Cruz, T.F., 1996. Reactive oxygen species mediate cytokine activation of c-Jun NH2-terminal kinases. J. Biol. Chem. 271, 15703–15707. Lupien, S.J., de Leon, M., de Santi, S., Convit, A., Tarshish, C., Nair, N.P.,Thakur, M., McEwen, B.S., Hauger, R.L., Meaney, M.J., 1998. Cortisol levels during human aging predict hippocampal atrophy and memory deficits. Nat. Neurosci. 1, 69–73. Luzzatto, L., Mehta, A., Vulliamy, T., 2001. Glucose-6-phosphate dehydrogenase deficiency. In: Scrivner, C.R., Baudet, A.L., Sly, W.S.,Valle,D. (Eds.), The Metabolic and Molecular Basis of Inherited Disease. McGraw-Hill, New York, pp. 4517–4553. Marks, P.A., Banks, J., 1960. Inhibition of mammalian glucose-6-phosphate dehydrogenase by steroids. Proc. Natl. Acad. Sci. U.S.A. 46, 447–452. Marletta, M.A., 1994. Nitric acid synthase: aspects concerning structure and catalysis. Cell 78, 927–930. Marnett, L.J., 2000. Oxyradicals and DNA damage. Carcinogenesis 21, 361–370. Marumo, T., Schini-Kerth, V.B., Fisslthaler, B., Busse, R., 1997. Platelet-derived growth factor-stimulated superoxide anion production modulates activation of transcription factor NF-kappaB and of monocyte chemoattractant protein 1 in human aortic smooth muscle. Circulation 96, 2361–2367. Masuzaki, H., Paterson, J., Shinyama, H., Morton, N.M., Mullins, J.J., Seckl, J.R., Flier, J.S., 2001. A transgenic model of visceral obesity and the metabolic syndrome. Science 294, 2166–2170. Miller, J.A., Miller, E.C., 1977. Ultimate chemical carcinogens as reactive mutagenic electrophiles. In: Hiatt, H.H., Watson, J.D., Winsten, J.A. (Eds.), Origins of Human Cancer. Cold Spring Harbor Laboratory, pp. 605–627. Misra, H.P., Gorsky, L.D., 1981. Paraquat and NADPH-dependent lipid peroxidation in lung microsomes. J. Biol. Chem. 256, 9994–9998. Mortola, J.F., Yen, S.S.C., 1990. The effects of oral dehydroepiandrosterone on endocrine-metabolic parameters in postmenopausal women. J. Clin. Endocrinol. Metab. 71, 696–704. Murray, C.J., Lopez, A.D., 1997. Global mortality, disability, and the contribution of risk factors: Global Burden of Disease Study. Lancet 349, 1436–1442. Nowicki, A., Szenajch, J., Ostrowska, G., Wojtowicz, A., Wojtowicz, K., Kruszewski, A.A., Maruszynski, M., Aukerman, S.L., Wiktor-Jedrzejczak, W., 1996. Impaired tumor growth in colony-stimulating factor 1 (CSF-1)-deficient, macrophage-deficient op/op mouse: evidence for a role of CSF-1-dependent macrophages in formation of tumor stroma. Int. J. Cancer 65, 112–119. A.G. Schwartz, L.L. Pashko / Ageing Research Reviews 3 (2004) 171–187 185

Nyce, J.W., Magee, P.N., Hard, G.C., Schwartz, A.G., 1984. Inhibition of 1,2-dimethylhydrazine-induced colon tumorigenesis in Balb/c mice by dehydroepiandrosterone. Carcinogenesis 5, 57–62. Olshansky, S.J., Hayflick, L., Carnes, B.A., 2002. Position statement on human aging. J. Gerontol. Biol. Sci. 57A, B292–B297. Orentreich, N., Brind, J.L., Rizer, R.L., 1984. Age changes and sex differences in serum dehydroepiandrosterone sulfate concentrations throughout adulthood. J. Clin. Endocrinol. Metab. 59, 551–555. Ozaki, M., Deshpande, S.S., Angkeow, P., Bellan, J., Lowenstein, C.J., Dinauer, M.C., Goldschmidt-Clermont, P.J., Irani, K., 2000. Inhibition of the Rac1 GTPase protects against nonlethal ischemia/reperfusion-induced necrosis and apoptosis in vivo. FASEB J. 14, 418–429. Pallman, J., Ackerman, L.J., 1996. 28-Day Oral Toxicity Study in Rats. Pharmakon USA. Parker Jr., C.R., 1999. Dehydroepiandrosterone and dehydroepiandrosterone sulfate production in the human adrenal during development and aging. Steroids 64, 640–647. Pascale, R., Garcea, R., Ruggiu, M.E., Daino, L., Frassetto, S., Vannini, M.G., Cozzolino, P., Lenzerini, L., Feo, F., Schwartz, A.G., 1987. Decreased stimulation by 12-O-tetradecanoylphorbol-13-acetate of superoxide radical production by polymorphonuclear leukocytes carrying the Mediterranean variant of glucose-6-phosphate dehydrogenase. Carcinogenesis 8, 1567–1570. Pashko, L.L., Rovito, R.J., Williams, J.R., Sobel, E.L., Schwartz, A.G., 1984. Dehydroepiandrosterone (DHEA) and 3␤-methylandrost-5-en-17-one: inhibitors of 7,12-dimethylbenz(a)anthracene (DMBA)-initiated and 12-O-tetradecanoylphorbol-13-acetate (TPA)-promoted skin papilloma formation in mice. Carcinogenesis 5, 463–466. Pashko, L.L., Hard, G.C., Rovito, R.J., Williams, J.R., Sobel, E.L., Schwartz, A.G., 1985. Dehydroepiandrosterone and 3␤-methylandrost-5-en-17-one inhibit 7,12-dimethylbenz(a)anthracene-induced skin papillomas and carcinomas in mice. Cancer Res. 45, 164–166. Pashko, L.L., Lewbart, M.L., Schwartz, A.G., 1991. Inhibition of 12-O-tetradecanoylphorbol-13-acetate-promoted skin tumor formation in mice by 16␣-fluoro-5-androsten-17-one and its reversal by deoxyribonucleosides. Carcinogenesis 12, 2189–2192. Pashko, L.L., Schwartz, A.G., 1993. Antihyperglycemic effect of the dehydroepiandrosterone analogue 16␣-fluoro-5-androsten-17-one in diabetic mice. Diabetes 42, 1105–1108. Passarino, G., Underhill, P.A., Cavalli-Sforza, L.L., Semino, O., Pes, G.M., Carru, C., Ferrucci, L., Bonafe, M., Franceschi, C., Deiana, L., Baggio, G., De Benedictis, G., 2001. Y chromosome binary markers to study the high prevalence of males in Sardinian centenarians and the genetic structure of the Sardinian population. Hum. Hered. 52, 136–139. Pecke, P.M., Chrousos, G.P., 1995. Hypercortisolism and obesity. Ann. N. Y. Acad. Sci. 771, 665–676. Perkins, S.N., Hursting, S.D., Haines, D.C., James, S.J., Miller, B.J., Phang, J.M., 1997. Chemoprevention of spontaneous tumorigenesis in nullizygous p53-deficient mice by dehydroepiandrosterone and its analog, 16␣-fluoro-5-androsten-17-one. Carcinogenesis 18, 989–994. Poole, J.C.E., Florey, H.W., 1958. Changes in the endothelium of the aorta and the behavior of macrophages in experimental atheroma of rabbits. J. Pathol. Bacteriol. 75, 245–253. Raineri, R., Levy, H.R., 1970. On the specificity of steroid interaction with mammary gland glucose-6-phosphate dehydrogenase. Biochemistry 9, 2233–2243. Rao, K.V., Johnson, W.D., Bosland, M.C., Lubet, R.A., Steele, V.E., Kelloff, G.J., McCormick, D.L., 1999. Chemoprevention of rat prostate carcinogenesis by early and delayed administration of dehydroepiandrosterone. Cancer Res. 59, 3084–3089. Rao, M.S., Subbarao, V., Yeldani, A.V., Reddy, J.K., 1992. Inhibition of spontaneous testicular Leydig cell tumor development in F-344 rats by dehydroepiandrosterone. Cancer Lett. 65, 123–126. Rao, M.S., Subbarao, V., Yeldani, A.V., Reddy, J.K., 1992. Hepatocarcinogenicity of dehydroepiandrosterone in the rat. Cancer Res. 52, 2977–2979. Ratko, T.A., Detrisac, C.J., Mehta, R.G., Kelloff, G.J., Moon, R.C., 1991. Inhibition of rat mammary gland chemical carcinogenesis by dietary dehydroepiandrosterone or a fluorinated analogue of dehydroepiandrosterone. Cancer Res. 51, 481–486. Roth, G.S., Lane, M.A., Ingram, D.K., Mattison, J.A., Elahi, D., Tobin, J.D., Muller, D., Metter, E.J., 2002. Biomarkers of caloric restriction may predict longevity in humans. Science 297, 811. Sadowski, I.J., Wright, J.A., Israels, L.G., 1985. A permeabilized cell system for studying regulation of aryl hydrocarbon hydroxylase: NADPH as rate limiting factor in benzo[a]pyrene metabolism. Int. J. Biochem. 17, 1023–1025. 186 A.G. Schwartz, L.L. Pashko / Ageing Research Reviews 3 (2004) 171–187

Sapolsky, R.M., 1985. A mechanism for glucocorticoid toxicity in the hippocampus: increased neuronal vulnerability to metabolic insults. J. Neurosci. 5, 1228–1232. Schachter, F., Faure-Delanef, L., Guenot, F., Rouger, H., Froguel, P., Lesueur-Ginot, L., Cohen, D., 1994. Genetic associations with human longevity at the APOE and ACE loci. Nat. Genet. 6, 29–32. Schreck, R., Rieber, P., Baeuerle, P.A., 1991. Reactive oxygen intermediates as apparently widely used messengers in activation of NF-␬B transcription factor and HIV-1. EMBO J. 10, 2247–2258. Schwartz, A.G., Perantoni, A., 1975. Protective effect of dehydroepiandrosterone against aflatoxin B1- and 7,12-dimethylbenz(a)anthracene-induced cytotoxicity and transformation in cultured cells. Cancer Res. 35, 2482–2487. Schwartz, A.G., 1979. Inhibition of spontaneous breast cancer formation in female C3H-Avy/A mice by long-term treatment with dehydroepiandrosterone. Cancer Res. 39, 1129–1132. Schwartz, A.G., Tannen, R.H., 1981. Inhibition of 7,12-dimethylbenz(a)anthracene- and urethan-induced lung tumor formation in A/J mice by long-term treatment with dehydroepiandrosterone. Carcinogenesis 2, 1335– 1338. Schwartz, A.G., Lewbart, M.L., Pashko, L.L., 1988. Novel dehydroepiandrosterone analogues with enhanced biological activity and reduced side-effects in mice and rats. Cancer Res. 48, 4817–4822. Schwartz, A.G., Fairman, D.K., Polansky, M., Lewbart, M.L., Pashko, L.L., 1989. Inhibition of 7,12-dimethylbenz(a)anthracene-initiated and tetradecanoylphorbol-13-acetate-promoted skin papilloma formation in mice by dehydroepiandrosterone and two synthetic analogs. Carcinogenesis 10, 1809–1813. Schwartz, A.G., Pashko, L.L., 1993. Cancer chemoprevention with the adrenocortical steroid dehydroepiandrosterone and structural analogs. J. Cell. Biochem. 17G, 73–79. Schwartz, A.G., Pashko, L.L., 2001. Suppression of 12-O-tetradecanoylphorbol-13-acetate-induced epidermal hyperplasia and inflammation by the dehydroepiandrosterone analog 16␣-fluoro-5-androsten-17-one and its reversal by NADPH–liposomes. Cancer Lett. 168, 7–14. Shantz, L.M., Talalay, P., Gordon, G.B., 1989. Mechanism of inhibition of growth of 3T3-L1 fibroblasts and their differentiation to adipocytes by dehydroepiandrosterone and related steroids: role of glucose-6-phosphate dehydrogenase. Proc. Natl. Acad. Sci. U.S.A. 86, 3852–3856. Simile, M., Pascale, R.M., DeMiglio, M.R., Nufris, A., Daino, L., Seddaiu, M.A., Muroni, M.R., Rao, K.N., Feo, F., 1995. Inhibition by dehydroepiandrosterone of growth and progression of persistent liver nodules in experimental rat liver carcinogenesis. Int. J. Cancer 62, 210–215. Siniscalco, M., Bernini, L., Latte, B., Motulsky, A.G., 1961. Favism and thalassemia in Sardinia and their relationship to malaria. Nature 190, 1179–1180. Steinberg, D., 2002. Atherogenesis in perspective: hypercholesteremia and inflammation as partners in crime. Nat. Med. 8, 1211–1217. Suh, Y.-A., Arnold, R.S., Lasegue, B., Shih, J., Xu, X., Sorescu, D., Chung, A.B., Griendling, K.K., Lambeth, J.D., 1999. Cell transformation by the superoxide-generating oxidase, MOX 1. Nature 401, 79–82. Sundaresan, M., Zu-Xi, Y., Ferrans, V.J., Irani, K., Finkel, T., 1995. Requirement for generation of H2O2 for platelet-derived growth factor signal transduction. Science 270, 296–299. Svec, F., Porter, J.F., 1998. The actions of exogenous dehydroepiandrosterone in experimental animals and humans. Proc. Soc. Exp. Biol. Med. 218, 174–191. Thomas, T., Thomas, G., McLendon, C., SuHon, T., Mullan, M., 1996. ␤-Amyloid-mediated vasoactivity and vascular endothelial damage. Nature 380, 168–171. Tian, W.-N., Braunstein, L.D., Pang, J., Stuhlmeier, K.M., Xi, Q.C., Tian, S., Stanton, R.C., 1998. Importance of glucose-6-phosphate dehydrogenase activity for cell growth. J. Biol. Chem. 273, 10609–10617. Van der Wal, A.C., Becker, A.E., van der Loos, C.M., Das, P.K., 1994. Site of intimal rupture or erosion of thrombosed coronary atherosclerotic plaques is characterized by an inflammatory process irrespective of the dominant plaque morphology. Circulation 89, 36–44. Vulliamy, T.J., D’Urso, M., Battistuzzi, G., Estrada, M., Foulkes, N.S., Martini, G., Calabro, V., Poggi, V., Giordano, R., Town, M., Luzzato, L., Persico, M.G., 1988. Diverse point mutations in the human glucose-6-phosphate dehydrogenase gene cause enzyme deficiency and mild or severe hemolytic anemia. Proc. Natl. Acad. Sci. U.S.A. 85, 5171–5175. Vulliamy, T., Mason, P., Luzzatto, L., 1992. The molecular basis of glucose-6-phosphate dehydrogenase deficiency. Trends Genet. 8, 138–143. A.G. Schwartz, L.L. Pashko / Ageing Research Reviews 3 (2004) 171–187 187

Walder, C.E., Green, S.P., Darbonne, W.C., Mathias, J., Rae, J., Dinauer, M.C., Curnutte, J.T., Thomas, G.R., 1997. Ischemic stroke injury is reduced in mice lacking a functional NADPH oxidase. Stroke 28, 2252–2258. Whitcomb, J.M., Schwartz, A.G., 1985. Dehydroepiandrosterone and 16␣-Br epiandrosterone inhibit 12-O-tetradecanoylphorbol-13-acetate stimulation of superoxide radical production by human polymorphonuclear leukocytes. Carcinogenesis 6, 333–335.