Benson T. Akingbemi

Why public health agencies cannot depend on good laboratory practices as a criterion for selecting data: The case of Bisphenol A

Background In their safety evaluations of bisphenol A (BPA), the U.S. Food and Drug Administration (FDA) and a counterpart in Europe, the European Food Safety Authority (EFSA), have given special prominence to two industry-funded studies that adhered to standards defined by Good Laboratory Practices (GLP). These same agencies have given much less weight in risk assessments to a large number of independently replicated non-GLP studies conducted with government funding by the leading experts in various fields of science from around the world. Objectives We reviewed differences between industry-funded GLP studies of BPA conducted by commercial laboratories for regulatory purposes and non-GLP studies conducted in academic and government laboratories to identify hazards and molecular mechanisms mediating adverse effects. We examined the methods and results in the GLP studies that were pivotal in the draft decision of the U.S. FDA declaring BPA safe in relation to findings from studies that were competitive for U.S. National Institutes of Health (NIH) funding, peer-reviewed for publication in leading journals, subject to independent replication, but rejected by the U.S. FDA for regulatory purposes. Discussion Although the U.S. FDA and EFSA have deemed two industry-funded GLP studies of BPA to be superior to hundreds of studies funded by the U.S. NIH and NIH counterparts in other countries, the GLP studies on which the agencies based their decisions have serious conceptual and methodologic flaws. In addition, the U.S. FDA and EFSA have mistakenly assumed that GLP yields valid and reliable scientific findings (i.e., “good science”). Their rationale for favoring GLP studies over hundreds of publically funded studies ignores the central factor in determining the reliability and validity of scientific findings, namely, independent replication, and use of the most appropriate and sensitive state-of-the-art assays, neither of which is an expectation of industry-funded GLP research. Conclusions Public health decisions should be based on studies using appropriate protocols with appropriate controls and the most sensitive assays, not GLP. Relevant NIH-funded research using state-of-the-art techniques should play a prominent role in safety evaluations of chemicals.

Estrogen regulation of testicular function

Evidence supporting a role for estrogen in male reproductive tract development and function has been collected from rodents and humans. These studies fall into three categories: i) localization of aromatase and the target protein for estrogen (ER-alpha and ER-beta) in tissues of the reproductive tract; ii) analysis of testicular phenotypes in transgenic mice deficient in aromatase, ER-alpha and/or ER-beta gene; and, iii) investigation of the effects of environmental chemicals on male reproduction. Estrogen is thought to have a regulatory role in the testis because estrogen biosynthesis occurs in testicular cells and the absence of ERs caused adverse effects on spermatogenesis and steroidogenesis. Moreover, several chemicals that are present in the environment, designated xenoestrogens because they have the ability to bind and activate ERs, are known to affect testicular gene expression. However, studies of estrogen action are confounded by a number of factors, including the inability to dissociate estrogen-induced activity in the hypothalamus and pituitary from action occurring directly in the testis and expression of more than one ER subtype in estrogen-sensitive tissues. Use of tissue-specific knockout animals and administration of antiestrogens and/or aromatase inhibitors in vivo may generate additional data to advance our understanding of estrogen and estrogen receptor biology in the developing and mature testis.

The Industrial Chemical Bisphenol A (BPA) Interferes with Proliferative Activity and Development of Steroidogenic Capacity in Rat Leydig Cells

ABSTRACT The presence of bisphenol A (BPA) in consumer products has raised concerns about potential adverse effects on reproductive health. Testicular Leydig cells are the predominant source of the male sex steroid hormone testosterone, which supports the male phenotype. The present report describes the effects of developmental exposure of male rats to BPA by gavage of pregnant and lactating Long-Evans dams at 2.5 and 25 μg/kg body weight from Gestational Day 12 to Day 21 postpartum. This exposure paradigm stimulated Leydig cell division in the prepubertal period and increased Leydig cell numbers in the testes of adult male rats at 90 days. Observations from in vitro experiments confirmed that BPA acts directly as a mitogen in Leydig cells. However, BPA-induced proliferative activity in vivo is possibly mediated by several factors, such as 1) protein kinases (e.g., mitogen-activated protein kinases or MAPK), 2) growth factor receptors (e.g., insulin-like growth factor 1 receptor-beta and epidermal growth factor receptors), and 3) the Sertoli cell-secreted anti-Mullerian hormone (also called Mullerian inhibiting substance). On the other hand, BPA suppressed protein expression of the luteinizing hormone receptor (LHCGR) and the 17beta-hydroxysteroid dehydrogenase enzyme (HSD17B3), thereby decreasing androgen secretion by Leydig cells. We interpret these findings to mean that the likely impact of deficits in androgen secretion on serum androgen levels following developmental exposure to BPA is alleviated by increased Leydig cell numbers. Nevertheless, the present results reinforce the view that BPA causes biological effects at environmentally relevant exposure levels and its presence in consumer products potentially has implication for public health.

Flawed Experimental Design Reveals the Need for Guidelines Requiring Appropriate Positive Controls in Endocrine Disruption Research

Frederick S. vom Saal,* Benson T. Akingbemi,† Scott M. Belcher,‡ David A. Crain,§ David Crews,{ Linda C. Guidice,jj Patricia A. Hunt,jjj Csaba Leranth,jjjj John Peterson Myers,# Angel Nadal,** Nicholas Olea,†† Vasantha Padmanabhan, Cheryl S. Rosenfeld, Alan Schneyer, Gilbert Schoenfelder, Carlos Sonnenschein, Ana M. Soto, Richard W. Stahlhut, Shanna H. Swan, Laura N. Vandenberg, Hong-Sheng Wang, Cheryl S. Watson, Wade V. Welshons, and Robert T. Zoeller

Developmental Exposures of Male Rats to Soy Isoflavones Impact Leydig Cell Differentiation

Testicular Leydig cells, which are the predominant source of the male sex steroid hormone testosterone, express estrogen receptors (ESRs) and are subject to regulation by estrogen. Following ingestion, the two major isoflavones in soybeans, genistin and daidzin, are hydrolyzed by gut microflora to form genistein and daidzein, which have the capacity to bind ESRs and affect gene expression. Thus, the increasing use of soy-based products as nondairy sources of protein has raised concerns about the potential of these products to cause reproductive toxicity. In the present study, perinatal exposure of male rats to isoflavones induced proliferative activity in Leydig cells. Isoflavones have the capacity to act directly as mitogens in Leydig cells, because genistein treatment induced Leydig cell division in vitro. Genistein action regulating Leydig cell division involved ESRs, acting in concert with signaling molecules in the transduction pathway mediated by protein kinase B (AKT) and mitogen-activated protein kinase (MAPK). Enhanced proliferative activity in the prepubertal period increased Leydig cell numbers, which alleviated deficits in androgen biosynthesis and/or augmented serum and testicular testosterone concentrations in adulthood. Together, these observations indicate that the perinatal exposures of male rats to isoflavones affected Leydig cell differentiation, and they imply that including soy products in the diets of neonates has potential implications for testis function.

Effects of genistein and equol on human and rat testicular 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase 3 activities

The objective of the present study was to investigate the effects of genistein and equol on 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and 17beta-hydroxysteroid dehydrogenase 3 (17beta-HSD3) in human and rat testis microsomes. These enzymes (3beta-HSD and 17beta-HSD3), along with two others (cytochrome P450 side-chain cleavage enzyme and cytochrome P450 17alpha-hydroxylase/17-20 lyase), catalyze the reactions that convert the steroid cholesterol into the sex hormone testosterone. Genistein inhibited 3beta-HSD activity (0.2 micromol L(-1) pregnenolone) with half-maximal inhibition or a half-maximal inhibitory concentration (IC(50)) of 87 +/- 15 (human) and 636 +/- 155 nmol L(-1) (rat). Genisteins mode of action on 3beta-HSD activity was competitive for the substrate pregnenolonrge and noncompetitive for the cofactor NAD(+). There was no difference in genisteins potency of 3beta-HSD inhibition between intact rat Leydig cells and testis microsomes. In contrast to its potent inhibition of 3beta-HSD, genistein had lesser effects on human and rat 17beta-HSD3 (0.1 micromol L(-1) androstenedione), with an IC(50) >or= 100 micromol L(-1). On the other hand, equol only inhibited human 3beta-HSD by 42%, and had no effect on 3beta-HSD and 17beta-HSD3 in rat tissues. These observations imply that the ability of soy isoflavones to regulate androgen biosynthesis in Leydig cells is due in part to action on Leydig cell 3beta-HSD activity. Given the increasing intake of soy-based food products and their potential effect on blood androgen levels, these findings are greatly relevant to public health.

Inhibition of human and rat 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase 3 activities by perfluoroalkylated substances☆

Perfluoroalkylated substances (PFASs) including perfluorooctane acid (PFOA) and perfluorooctane sulfonate (PFOS) have been classified as persistent organic pollutants and are known to cause reduced testosterone production in human males. The objective of the present study was to compare the potencies of five different PFASs including PFOA, PFOS, potassium perfluorooctane sulfonate (PFOSK), potassium perfluorohexane sulfonate (PFHxSK) and potassium perfluorobutane sulfonate (PFBSK) in the inhibition of 3beta-hydroxysteroid dehydrogenase (3beta-HSD) and 17beta-hydroxysteroid dehydrogenase 3 (17beta-HSD3) activities in the human and rat testes. Human and rat microsomal enzymes were exposed to various PFASs. PFOS and PFOSK inhibited rat 3beta-HSD activity with IC(50) of 1.35 + or -0.05 and 1.77 + or - 0.04 microM, respectively, whereas PFHxSK and PFBSK had no effect at concentrations up to 250 microM. All chemicals tested weakly inhibited human 3beta-HSD activity with IC(50)s over 250 microM. On the other hand, PFOS, PFOSK and PFOA inhibited human 17beta-HSD3 activity with IC(50)s of 6.02 + or - 1.02, 4.39 + or - 0.46 and 127.60 + or - 28.52 microM, respectively. The potencies for inhibition of 17beta-HSD3 activity were determined to be PFOSK>PFOS>PFOA>PFHxSK=PFBSK for human 17beta-HSD3 activity. There appears to be a species-dependent sensitivity to PFAS-mediated inhibition of enzyme activity because the IC(50)s of PFOS(K) for inhibition of rat 17beta-HSD3 activity was greater than 250 microM. In conclusion, the present study shows that PFOS and PFOSK are potent inhibitors of rat 3beta-HSD and human 17beta-HSD3 activity, and implies that inhibition of steroidogenic enzyme activity may be a contributing factor to the effects that PFASs exert on androgen secretion in the testis.

Thiazolidinediones/PPARγ agonists and fatty acid synthase inhibitors as an experimental combination therapy for prostate cancer

The prostate cancer (PCa) cell lines LNCaP, PC-3, and DU-145 express peroxisome proliferator-activated receptor γ (PPARγ) but its role in PCa is unclear. Thiazolidinediones (TZDs), a family of PPARγ activators and type 2 anti-diabetic drugs, exhibit anti-tumor apoptotic effects in human PCa cell lines. Likewise, pharmacological inhibitors of fatty acid synthase (FASN), a metabolic enzyme highly expressed in PCa, induce apoptosis in prostate and other cancer cells. Here, we show positive correlation between PPARγ and FASN protein in PCa cell lines and synergism between TZDs and FASN blockers in PCa cell viability reduction and apoptosis induction. Combined TZDs/FASN has enhanced anti-tumor properties in both androgen-dependent LNCaP and androgen-independent PC-3 and DU-145 cells when compared with single drug exposure. Low concentrations (5-10 μM) of the TZD drug rosiglitazone failed to alter cell viability but, paradoxically, upregulated lipogenic genes [PPARγ, FASN, sterol regulatory element binding protein-1c (SREBP-1c) and acetyl-Co A carboxylase-1 (ACC1)], which diminish the apoptotic effects of rosiglitazone. The mean IC50 in all cell lines was 45 ± 2 μM for rosiglitazone compared with significantly lower 5 ± 1 μM for rosiglitazone plus the FASN blocker cerulenin, and 10.2 ± 2 μM for rosiglitazone plus the cerulenin synthetic analog C75. The IC50 for the combined rosiglitazone and FASN blockers contrasts with the relatively higher IC50 for rosiglitazone (45 ± 2 μM), the TZD drug troglitazone (13 ± 2 μM), cerulenin (32 ± 1 μM), or C75 (26 ± 3 μM) when these drugs were used alone. In summary, this study shows proof-of-principle for combining FASN blockers and TZDs for PCa treatment.

Effects of phthalates on 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase 3 activities in human and rat testes.

The 3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase 3 (17β-HSD3) are involved in the reactions that culminate in androgen biosynthesis in Leydig cells. Human and rat testis microsomes were used to investigate the inhibitory potencies on 3β-HSD and 17β-HSD3 activities of 14 different phthalates with various carbon numbers in the ethanol moiety. The results demonstrated that the half-maximal inhibitory concentrations (IC(50)s) of dipropyl (DPrP), dibutyl (DBP), dipentyl (DPP), bis(2-butoxyethyl) (BBOP) and dicyclohexyl (DCHP) phthalate were 123.0, 24.1, 25.5, 50.3 and 25.5μM for human 3β-HSD activity, and 62.7, 30.3, 33.8, 82.6 and 24.7μM for rat 3β-HSD activity, respectively. However, only BBOP and DCHP potently inhibited human (IC(50)s, 23.3 and 8.2μM) and rat (IC(50)s, 30.24 and 9.1μM) 17β-HSD3 activity. Phthalates with 1-2 or 7-8 carbon atoms in ethanol moieties had no effects on both enzyme activities even at concentrations up to 1mM. The mode of action of DCHP on 3β-HSD activity was competitive with the substrate pregnenolone but noncompetitive with the cofactor NAD+. The mode of action of DCHP on 17β-HSD3 activity was competitive with the substrate androstenedione but noncompetitive with the cofactor NADPH. In summary, our results showed that there are clear structure-activity responses for phthalates in the inhibition of both 3β-HSD and 17β-HSD3 activities. The length of carbon chains in the ethanol moieties of phthalates may determine the potency to inhibit these two enzymes.

Aflatoxin B1 disrupts the androgen biosynthetic pathway in rat Leydig cells.

The present study investigated if Aflatoxin B1 (AFB1), a potent and naturally occurring mycotoxin, interferes with the steroidogenic pathway in rat Leydig cells. Testicular Leydig cells are the predominant source of the male sex steroid hormone testosterone (T) that maintains the male phenotype and support fertility. Leydig cells, isolated from 35-day-old male Long-Evans rats (Rattus norvegicus), were incubated with AFB1 at 0, 0.01, 0.1, 1, 10μM followed by measurement of T secretion by radioimmunoassay and analysis of protein expression in western blots. Results demonstrated that AFB1 suppressed testosterone secretion in a dose-dependent manner and inhibited expression of cholesterol transporter steroidogenic acute regulatory protein (StAR) and steroidogenic enzymes [(3β-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase enzyme (HSD17B3)]. Protein expression analysis showed that AFB1 treatment increased ERK phosphorylation but suppressed p38 MAPK and JNK activation in Leydig cells. AFB1-induced inhibition of Leydig cells was alleviated by co-treatment with the ERK inhibitor UO 126, implying that ERK mediates, at least in part, the inhibitory effects of AFB1 in Leydig cells. The findings highlight potential extra-hepatic effects of aflatoxin exposure and indicate that exposure to AFB1 has significant reproductive health implications for consumers of contaminated products even under conditions of low dietary toxin levels.