Good Laboratory Practices: Myers et al. Respond

Reproduced with permission from Environmental Health Perspectives. DOI:10.1289/ehp.0900884RMyers et al. respond to a letter written by Becker et al. regarding Myers' article "Why public health agencies cannot depend on Good Laboratory Practices as a criterion for selecting data: the case of bisphenol A.

Estrogen regulation of testicular function

Abstract 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.</p

Inhibition of testicular steroidogenesis by the xenoestrogen bisphenol A is associated with reduced pituitary luteinizing hormone secretion and decreased steroidogenic enzyme gene expression in rat Leydig cells

Exposure of humans to bisphenol A (BPA), a monomer in polycarbonate plastics and a constituent of resins used in food packaging and dentistry, is significant. In this report exposure of rats to 2.4 μg/kg·d (a dose that approximates BPA levels in the environment) from postnatal d 21-35 suppressed serum LH (0.21 ± 0.05 ng/ml; vs. control, 0.52 ± 0.04; P \u3c 0.01) and testosterone (T) levels (1.62 ± 0.16 ng/ml; vs. control, 2.52 ± 0.21; P \u3c 0.05), in association with decreased LHβ and increased estrogen receptor β pituitary mRNA levels as measured by RT-PCR. Treatment of adult Leydig cells with 0.01 nM BPA decreased T biosynthesis by 25% as a result of decreased expression of the steroidogenic enzyme 17α-hydroxylase/ 17-20 lyase. BPA decreased serum 17β-estradiol levels from 0.31 ± 0.02 ng/ml (control) to 0.22 ± 0.02, 0.19 ± 0.02, and 0.23 ± 0.03 ng/ml in rats exposed to 2.4 μg, 10 μg, or 100 mg/kg·d BPA, respectively, from 21-35 d of age (P \u3c 0.05) due to its ability to inhibit Leydig cell aromatase activity. Exposures of pregnant and nursing dams, i.e. from gestation d 12 to postnatal d 21, decreased T levels in the testicular interstitial fluid from 420 ± 34 (control) to 261 ± 22 (P \u3c 0.05) ng/ml in adulthood, implying that the perinatal period is a sensitive window of exposure to BPA. As BPA has been measured in several human populations, further studies are warranted to assess the effects of BPA on male fertility

Effects of methoxychlor and 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane on 3β-hydroxysteroid dehydrogenase and 17β-hydroxysteroid dehydrogenase-3 activities in human and rat testes

Human and rat testis microsomes were used to investigate direct inhibitory activities of methoxychlor (MXC) and its metabolite 2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane (HPTE) on 3ß-hydroxysteroid dehydrogenase (3β-HSD) and 17β-hydroxysteroid dehydrogenase type 3 (17β-HSD3). The 3β-HSD and 17β-HSD3 enzymes are involved in the reactions that culminate in androgen biosynthesis in Leydig cells. The results demonstrated that MXC and HPTE inhibited human 3ß-HSD activity at a concentration of 10 nm. The half maximal inhibitory concentration (IC_50) for MXC inhibition of 3β-HSD was 53.21 ± 15.52 µm (human) and 46.15 ± 17.94 µm (rat), and for HPTE, it was 8.29 ± 2.49 µm (human) and 13.82 ± 2.26 µm (rat). At the higher concentration of 100 µm, MXC did not affect human and rat 17β-HSD3 activity. However, the IC_50 for HPTE inhibition of 17β-HSD3 was 12.1 ± 1.9 µm (human) and 32 .0 ± 8.6 µm (rat). The mode of action of MXC and HPTE on 3β-HSD activity was non-competitive with the substrate pregnenolone, but was competitive with the cofactor NAD^+. The mode of HPTE inhibition of 17β-HSD3 was non-competitive with the substrate androstenedione, but was competitive with the cofactor NADPH. In summary, our results showed that HPTE, which is the biologically active metabolite of MXC, has the capacity for direct inhibition of 3β-HSD and 17β-HSD3 enzyme activity. Inhibition of enzyme activity is presumably associated with suppression of steroidogenesis in gonadal tissues and has implications for testis function

Biphasic effects of postnatal exposure to diethylhexylphthalate on the timing of puberty in male rats

Phthalate esters such as di(2-ethylhexyl)phthalate (DEHP), which are commonly found in cosmetics and in flexible plastics distributed by the food, construction, and medical products industries, have been classified as anti-androgens. High-dose DEHP exposure in utero is associated with decreased androgen levels. However, when administered after birth, low doses of DEHP (eg, 10 mg/kg body weight) may stimulate androgen production. In the present study, the potential of phthalate exposure to advance or delay the timing of puberty was assessed. Male Long-Evans rat pups were chronically subjected to low or high doses of DEHP, with the androgen-driven process of preputial separation serving as an index of pubertal timing. Rats were treated with 0, 10, 500, or 750 mg/kg body weight DEHP for 28 days starting at day 21 postpartum. The average age at which the animals completed preputial separation was measured in each group. The age of preputial separation was 41.5 ± 0.1 days postpartum in controls (vehicle). The 10 mg/kg DEHP dose advanced pubertal onset significantly to 39.7 ± 0.1 days postpartum, whereas the 750 mg/kg DEHP dose delayed pubertal onset to 46.3 ± 0.1 days postpartum. The 10 mg/kg DEHP dose also significantly increased serum testosterone (T) levels (3.13 ± 0.37 ng/mL) and seminal vesicle weights (0.33 ± 0.02 g) compared with control serum T (1.98 ± 0.20 ng/mL) and seminal vesicle weight (0.26 ± 0.02 g), while the 750 mg/kg dose decreased serum T (1.18 ± 0.18 ng/mL) as well as testes and body weights. Direct action of the DEHP metabolite, monoethylhexylphthalate (MEHP), on Leydig cell steroidogenic capacity was investigated in vitro. MEHP treatment at a low concentration (100 μM) increased luteinizing hormone-stimulated T production, whereas 10 mM concentrations were inhibitory. In conclusion, data from the present study indicate that DEHP has a biphasic effect on Leydig cell function, with low-dose exposure advancing the onset of puberty. High doses of DEHP, which are anti-androgenic, may also be outside the range of real environmental exposure levels