Annika Adamsson

Female field voles with high testosterone and glucose levels produce male-biased litters

The proximate physiological mechanisms producing the parental ability to vary offspring sex ratio in many vertebrates remain elusive. Recently, high concentrations of maternal testosterone and glucose and low concentrations of maternal corticosterone have been suggested to explain male bias in offspring sex ratio. We examined how these factors affect secondary offspring sex ratio in nondomesticated field voles, Microtus agrestis, while controlling for maternal age, testosterone level of the male and body condition of both the female and the male. We found that females with high preconception serum testosterone and glucose levels produced a male-biased litter, whereas there was no association between maternal corticosterone level and litter sex ratio. Older females produced a bias towards sons, but neither their body condition nor paternal testosterone level correlated with litter sex ratio. Finally, females mated with a high body-condition male tended to deliver a male-biased litter. Our results suggest that several physiological traits of the mother may simultaneously be related to offspring sex ratio in mammals.

Cryptorchidism and endocrine disrupting chemicals

Prospective clinical studies have suggested that the rate of congenital cryptorchidism has increased since the 1950s. It has been hypothesized that this may be related to environmental factors. Testicular descent occurs in two phases controlled by Leydig cell-derived hormones insulin-like peptide 3 (INSL3) and testosterone. Disorders in fetal androgen production/action or suppression of Insl3 are mechanisms causing cryptorchidism in rodents. In humans, prenatal exposure to potent estrogen diethylstilbestrol (DES) has been associated with increased risk of cryptorchidism. In addition, epidemiological studies have suggested that exposure to pesticides may also be associated with cryptorchidism. Some case-control studies analyzing environmental chemical levels in maternal breast milk samples have reported associations between cryptorchidism and chemical levels. Furthermore, it has been suggested that exposure levels of some chemicals may be associated with infant reproductive hormone levels.

Cadmium-induced effects on cellular signaling pathways in the liver of transgenic estrogen reporter mice.

Estrogen-like effects of cadmium (Cd) have been reported in several animal studies, and recent epidemiological findings suggest increased risk of hormone-dependent cancers after Cd exposure. The mechanisms underlying these effects are still under investigation. Our aim was to study the effects of Cd on cellular signaling pathways in vivo with special focus on estrogen signaling and to perform benchmark dose analysis on the effects. Transgenic adult ERE-luciferase male mice were exposed subcutaneously to 0.5-500 μg CdCl(2) per kg body weight (bw) or 17α-ethinylestradiol (EE2) for 3 days. These doses had no effects on organ and bw or testicular histology, indicating subtoxic exposure levels. The transgene luciferase, reporting genomic estrogen response, was significantly increased by EE2 but not by Cd. However, Cd significantly affected kinase phosphorylation and endogenous gene expression. Interestingly, gene expression changes displayed a traditional dose-response relationship, with benchmark dose levels for the expression of Mt1, Mt2, p53, c-fos, and Mdm2 being 92.9, 19.9, 7.6, 259, and 25.9 μg/kg bw, respectively, but changes in kinase phosphorylation were only detected at low exposure levels. Phosphorylation of Erk1/2 was significantly increased even in the lowest dose group, 0.5 μg/kg bw, rendering pErk1/2 a more sensitive sensor of exposure than changes in gene expression. Collectively, our data suggest that the effects triggered by Cd in vivo are markedly concentration dependent. Furthermore, we conclude that the estrogen-like effects of Cd are likely to result from a mechanism different from steroidal estrogens.

The effects of 2,3,7,8-tetrachlorodibenzo-p-dioxin on foetal male rat steroidogenesis

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most toxic and widely investigated dioxin congener. In utero and lactational exposure to TCDD results in developmental and reproductive defects that are the most sensitive endpoints for TCDD toxicity. TCDD has a potential to interfere with steroid metabolism, but the mechanisms by which this occurs are not well understood. In this study, we investigated the effects of TCDD on prenatal rat steroidogenesis. Pregnant Sprague-Dawley female rats were treated once with TCDD (0, 0.3 or 1 microg/kg) by gavage on embryonic day (ED) 11 and the expression levels of androgen (AR) and estrogen receptors (ER), steroidogenic enzymes (P450scc and 3beta-HSD) and four regulatory factors (StAR, SF-1, GATA-4 and Insl-3) involved in foetal Leydig cell and adrenal function were analysed on ED 19.5. Hormonal status of male foetuses was determined by measuring testicular testosterone (T) levels, plasma luteinizing hormone (LH) and corticosterone concentrations. In utero exposure to TCDD reduced intratesticular T of foetal males (significant at 0.3 microg/kg TCDD) and tended to reduce the protein expression of ERalpha and AR of foetal male rat testis. Foetal male rat plasma LH levels were significantly reduced at the dose of 1 microg/kg TCDD, while corticosterone levels tended to be increased possibly because of the TCDD-induced stress. Only minor alterations in steroidogenesis were observed in rat adrenal. mRNA expression of developmental regulatory factors was not influenced by foetal TCDD exposure, except for significantly reduced adrenal SF-1. The results demonstrate that maternal exposure to TCDD suppressed testicular steroidogenesis of 19.5-day-old foetal male Sprague-Dawley rat. The highest dose of TCDD (1 microg/kg) had also an effect on pituitary LH secretion. Our data implicate that TCDD has direct testicular and pituitary effects on foetal male rat but with different dose-responses. These changes can lead to impaired steroidogenesis and it may result in the maldevelopment of the testis and weaken masculinization.

Antiandrogen Exposure in Utero Disrupts Expression of Desert Hedgehog and Insulin-Like Factor 3 in the Developing Fetal Rat Testis

Testicular development is an androgen-dependent process, and fetal exposure to antiandrogens disrupts male sexual differentiation. A variety of testicular disorders may result from impaired development of fetal Leydig and Sertoli cells. We hypothesized that antiandrogenic exposure during fetal development interferes with desert hedgehog (Dhh) signaling in the testis and results in impaired Leydig cell differentiation. Fetal rats were exposed in utero to the antiandrogen flutamide from 10.5 d post conception (dpc) until they were killed or delivery. Fetal testes were isolated at different time points during gestation and gene expression levels of Dhh, patched-1 (Ptc1), steroidogenic factor 1 (Sf1), cytochrome P450 side-chain cleavage (P450scc), 3beta-hydroxysteroid dehydrogenase type 1 (Hsd3b1), and insulin-like factor 3 (Insl3) were analyzed. To study direct effects of hedgehog signaling on testicular development, testes from 14.5 dpc fetuses were cultured for 3 d in the presence of cyclopamine, sonic hedgehog, or vehicle, and gene expression levels and testosterone secretion were analyzed. Organ cultures were also analyzed histologically, and cleaved-caspase 3 immunohistochemistry was performed to assess apoptosis. In utero exposure to flutamide decreased expression levels of Dhh, Ptc1, Sf1, P450scc, Hsd3b1, and Insl3, particularly from 17.5 dpc onward. Inhibition of hedgehog signaling in testis cultures resulted in similar effects on gene expression levels. Apoptosis in Wolffian ducts was increased by cyclopamine compared with sonic hedgehog- or vehicle-treated cultures. We conclude that exposure to the antiandrogen flutamide interferes with Dhh signaling resulting in an impaired differentiation of the fetal Leydig cells and subsequently leading to abnormal testicular development and sexual differentiation.

Toxicological Profile of Ultrapure 2,2′,3,4,4′,5,5′-Heptachlorbiphenyl (PCB 180) in Adult Rats

PCB 180 is a persistent non-dioxin-like polychlorinated biphenyl (NDL-PCB) abundantly present in food and the environment. Risk characterization of NDL-PCBs is confounded by the presence of highly potent dioxin-like impurities. We used ultrapure PCB 180 to characterize its toxicity profile in a 28-day repeat dose toxicity study in young adult rats extended to cover endocrine and behavioral effects. Using a loading dose/maintenance dose regimen, groups of 5 males and 5 females were given total doses of 0, 3, 10, 30, 100, 300, 1000 or 1700 mg PCB 180/kg body weight by gavage. Dose-responses were analyzed using benchmark dose modeling based on dose and adipose tissue PCB concentrations. Body weight gain was retarded at 1700 mg/kg during loading dosing, but recovered thereafter. The most sensitive endpoint of toxicity that was used for risk characterization was altered open field behavior in females; i.e. increased activity and distance moved in the inner zone of an open field suggesting altered emotional responses to unfamiliar environment and impaired behavioral inhibition. Other dose-dependent changes included decreased serum thyroid hormones with associated histopathological changes, altered tissue retinoid levels, decreased hematocrit and hemoglobin, decreased follicle stimulating hormone and luteinizing hormone levels in males and increased expression of DNA damage markers in liver of females. Dose-dependent hypertrophy of zona fasciculata cells was observed in adrenals suggesting activation of cortex. There were gender differences in sensitivity and toxicity profiles were partly different in males and females. PCB 180 adipose tissue concentrations were clearly above the general human population levels, but close to the levels in highly exposed populations. The results demonstrate a distinct toxicological profile of PCB 180 with lack of dioxin-like properties required for assignment of WHO toxic equivalency factor. However, PCB 180 shares several toxicological targets with dioxin-like compounds emphasizing the potential for interactions.

Abstract 1321: Estrogen-like effects of cadmium in male mice and the involvement of MAPKs pathway

Cadmium is a ubiquitous toxicant of environmental concern, classified as a category 1 human carcinogen. Clear estrogen-like effects of cadmium have been reported in ovariectomized rats after a single dose. Recent epidemiological findings might suggest increased risk of hormone-related cancers such as cancer of the endometrium and breast. However, the specific molecular mechanism of action is unknown. Recently, we have shown that estrogen-like effects of cadmium in female mice do not appear to be mediated via the classical estrogen receptor transcriptional pathway. However, some estrogen like effects, possibly mediated via the non-classical estrogen signaling pathway were observed. The aim of the present study was to investigate the estrogen-like effects of cadmium in male mice. ERE-luc male mice were injected subcutaneously with CdCl 2 at 0.5, 5, 50 or 500 µg/kg b.w. or with 17α-ethinylestradiol (EE2) on three consecutive days. Testicular weights and histology, body and organ weight, Cd-tissue retention, activation of MAPK pathways and ERE dependent luciferase expression were investigated. Body weight and relative liver and kidney weights were not altered in any of the CdCl 2 or EE2 treated animals as compared to the control. Relative testis weights were significantly decreased in 0.5 and 50µg CdCl 2 /kg b.w. treated animals as compared to control. Testicular histology analysis showed no significant changes in the tubulus diameter and epithelial thickness at stage VII, however some apoptotic cells were observed in mice treated with CdCl 2 . No significant alterations in ERE mediated luciferase activity were detected in any of the CdCl 2 treated groups. Phosphorylation of Mdm2 was significantly increased at relatively low-dose of CdCl 2 (5µg/kg b.w.), whereas no significant changes in phosphorylation of Akt were observed in CdCl 2 or EE2 treated animals. Phosphorylation of Erk was found to be significantly increased after exposure to 0.5, 5 and 50µg of CdCl 2 /kg b.w. as compared to control. Gene expression of c-myc, c-fos, c-jun and p53 appeared to be decreased at relatively low dose of CdCl 2 (5µg/kg b.w). No significant changes were observed in the expression of HSP32, p38, SP1 or ER-alpha after CdCl 2 exposure. In conclusion the results suggest the involvement of MAPKs signaling pathways in cadmium induced effects in vivo after short term exposure at the doses investigated. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1321. doi:10.1158/1538-7445.AM2011-1321