In vivo kinetics and DNA-binding properties of the Ah receptor in the golden Syrian hamster

The in vivo long-term cytosolic-nuclear kinetics and DNA-binding properties of the Ah receptor were examined in liver from the golden Syrian hamster. For the kinetic studies, a dose of [3H]2,3,7,8-tetrachlorodibenzo-p-dioxin ([3H]TCDD) that has been previously shown to produce maximal and sustained hepatic enzyme induction without substantial toxicity was used. Following an intraperitoneal dose of 10 micrograms/kg of [3H]TCDD, occupied cytosolic receptor levels reached a peak within 8 h and then decreased rapidly to a level that was approximately 2% of the total receptor. Throughout the 35-day period, unoccupied cytosolic receptor represented from 65 to 80% of the total receptor content. At 8 h following dosing, less than 30% of the total amount of receptor was associated with the nuclear fraction; this percentage declined slowly to less than 5% of the total at Day 35. The half-life for the decline in detectable nuclear receptor levels was 13 days and was similar to the half-life for the decline in [3H]TCDD content of the whole liver, cytosol, and nuclear extract. The Ah receptor contained in hamster hepatic cytosol underwent a ligand-dependent transformation in vitro to two forms having affinity for DNA-Sepharose, one of which was isolated from nuclei of animals treated with [3H]TCDD in vivo. A comparison of the specific binding recovered following various analytical procedures revealed that the binding of [3H]TCDD to the form not found in nuclear extracts was more labile under certain experimental conditions. These studies indicate the heterogeneity of the Ah receptor in hamster hepatic cytosol and suggest that DNA binding in vitro and nuclear uptake in vivo occur through a ligand-dependent transformation process. The maintenance of maximal hepatic enzyme induction is, in part, a consequence of the sustained presence in the nucleus of only a small percentage of the total receptor content. The whole-tissue kinetics of TCDD appears to be a major factor regulating the long-term retention of the TCDD-receptor complex in the nucleus.     

Oxidative metabolites of diethylstilbestrol in the fetal Syrian golden hamster

14C-Diethylstilbestrol was administered orally, intraperitoneally, and intrafetally to 15-day pregnant hamsters at a dose of 20 mg/kg body weight, and the radioactivity was determined in the fetus, placenta, and maternal liver after 6 hours. Significant amounts of radioactivity were found in these tissues in every case, indicating maternal-fetal and fetal-maternal transfer of diethylstilbestrol. Part of the radioactivity found in the tissues could not be extracted even after excessive washing. This implied the presence of reactive metabolites. In the fetal and placental extracts, eight oxidative metabolites of diethylstilbestrol were identified by mass fragmentography as hydroxy- and methoxy-derivatives of diethylstilbestrol, pseudodiethylstilbestrol, and dienestrol. The presence of oxidative metabolites in the hamster fetus and the covalent binding to tissue macromolecules are possibly associated with the fetotoxic effects of diethylstilbestrol.     

Effect of pretreatment of male Syrian golden hamsters with 7,8-benzoflavone and with diethylstilbestrol on P-450 isoenzyme activities and on microsomal diethylstilbestrol metabolism

Combined treatment of male Syrian golden hamsters with the synthetic estrogen diethylstilbestrol (DES) and 7,8-benzoflavone (7,8-BF) gives rise to a high incidence of hepatocellular carcinomas, whereas no such tumors are formed with DES alone nor with 7,8-BF alone. To determine whether alterations in DES metabolism may account for the observed hepatocarcinogenicity, we have studied the effect of pretreatment with 7,8-BF alone, DES alone and 7,8-BF plus DES on the levels of hepatic P-450 and cytochrome b5, on the activities of various P-450 isoenzymes and on microsomal DES metabolism. Hepatic P-450 content was significantly increased after pretreatment with 7,8-BF and decreased after DES, while combined pretreatment led to levels similar to those in untreated control animals. Hepatic cytochrome b5 was also elevated in 7,8-BF-treated hamsters; DES pretreatment had no effect, and combined pretreatment led to a slight increase. Four different substrates were used to probe P-450 isoenzyme activity. Aryl hydrocarbon hydroxylase (AHH), 7-ethoxycoumarin-O-deethylase (ECOD), 7-ethoxyresorufin-O-deethylase (EROD) and 7-pentoxyresorufin-O-dealkylase (PROD) were all elevated after 7,8-BF-pretreatment, while DES led to a decrease in these activities with the exception of AHH, where a transient increase which was observed after 8 and 20 weeks of pretreatment was back to control levels after 32 weeks. Combined pretreatment with 7,8-BF and DES led to an intermediate response (slight increase) with AHH, EROD and PROD, but not with ECOD, where a full induction comparable with that observed after 7,8-BF alone was elicited. In spite of the modulation of enzyme levels and activities observed after the various pretreatments, the metabolism of DES in microsomes from pretreated animals was virtually identical with that from controls. Therefore it is concluded that modulation of hepatic DES metabolism is not the reason for the observed hepatotumorigenicity; instead, it is speculated that 7,8-BF is the carcinogenic agent in this tumor model, and DES may act as a promotor.     

Profiles of polycyclic aromatic hydrocarbon metabolites after treatment with various inducers of microsomal rat liver monoxygenases (author's transl)

The microsomal oxidation of 12 frequently occurring environmental polycyclic aromatic hydrocarbons after incubation with rat-liver microsomes has been studied and their metabolites characterized by means of gas-liquid chromatography/mass spectrometry. The method enables the detection and characterisation of phenols, diols, triols, and tetrols as trimethylsilyl ethers beside the original hydrocarbons. Moreover, the induction properties of some carcinogenic and non-carcinogenic hydrocarbons (benz[a]anthracene, pyrene, chrysene, benzo[a]-pyrene, benzo[e]pyrene, benzo[b]fluoranthene, benzo[j]fluoranthene, benzo[k]fluoranthene) have been studied. Except pyrene and benzo[e]pyrene, all compounds investigated significant but different induction factors. The relevance of the induction for an estimation of the biological effect of environmental polycyclic aromatic hydrocarbons is discussed.     

Apoptosis in the epididymal epithelium of adult male golden hamster exposed to diethylstilbestrol.

Apoptosis in the testis and prostate exposed to disrupters of endocrine function, including diethylstilbestrol (DES), during neonatal or postnatal periods has repeatedly been demonstrated, but not in the mature epididymis. We investigated the effects of DES, a potent and synthetic estrogen, on apoptosis in the adult. Adult male golden hamsters received an SC injection of DES and were then sacrificed to collect epididymides after 1, 4, or 7 days of treatment. A significant decrease in epididymal weight and an increase in apoptotic cells were shown on the first day after DES injection. Flow cytometry showed that DES treatment (1 mg/kg) for 1, 4, or 7 days induced significant apoptosis both in the caput and the cauda epididymides. Greater numbers of apoptotic cells were detected in the caput than in the cauda at a fixed time after DES treatment. Serum levels of testosterone decreased markedly within 24 hr after DES administration, reaching undetectable levels of 0.1 ng/ml at 4 days and thereafter. These results indicate that DES administration can increase epididymal apoptosis with a decrease in serum testosterone levels. Because DES used to be injected into domestic animals, adult males also have a chance to take this substance through food. Our study indicates that exposure to DES in adults is as toxic as that in the perinatal period.     

Androgen receptor immunoreactivity in the male and female Syrian hamster brain

To investigate potential mechanisms for sex differences in the physiologic response to androgens, the present study compared the hormonal regulation of intracellular androgen receptor partitioning and the distribution of androgen receptor immunoreactivity in select brain regions from male and female hamsters. Androgen receptors were visualized on coronal brain sections. Two weeks after castration, androgen receptor immunoreactivity filled the neuronal nuclei and cytoplasm in males and females. In gonad‐intact males and females, androgen receptor immunoreactivity was limited to the cell nucleus. Whereas exogenous dihydrotestosterone prevented cytoplasmic immunoreactivity, estrogen at physiologic levels did not. These results suggest that nuclear androgen receptor immunoreactivity in gonad‐intact females is maintained by endogenous androgens, and that androgens have the potential to influence neuronal activity in either sex. However, sex differences in the number and staining intensity of androgen‐responsive neurons were apparent in select brain regions. In the ventral premammillary nucleus, ventromedial nucleus of the hypothalamus, and medial amygdaloid nucleus, androgen receptor staining was similar in gonadectomized males and females. In the lateral septum, posteromedial bed nucleus of the stria terminalis (BNSTpm), and medial preoptic nucleus, the number of androgen receptor–immunoreactive neurons was significantly lower in females (p < .05). Moreover, the integrated optical density/cell in BNSTpm was significantly less in females (1.28 ± 0.3 units) than in males (2.21 ± 0.2 units; p < .05). These sex differences in the number and staining intensity of androgen‐responsive neurons may contribute to sex differences in the behavioral and neuroendocrine responses to androgens. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 359–370, 1999     

Effects of thyroidectomy on the Ah receptor and enzyme inducibility by 2,3,7,8-tetrachlorodibenzo-p-dioxin in the rat liver

Thyroidectomy of rats confers some protection, by an unknown mechanism, from the weight loss, immunotoxicity, and mortality induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Since at least some of the many effects of TCDD appear to be mediated by the Ah receptor, perhaps the thyroid plays a role in regulation of this receptor, thereby modifying the toxicity of TCDD. We tested this hypothesis by comparing TCDD-binding characteristics of the receptor and hepatic enzyme inducibility by TCDD (a receptor-mediated response) in thyroidectomized (ThX) and euthyroid rats. There were no significant differences in levels of TCDD binding in vitro in hepatic cytosol, in receptor affinity, nor in the molecular size of the TCDD-bound receptor in untreated ThX rats compared to controls fed ad libitum or pair-fed. Total hepatic cytochrome P-450 (P-450) levels and NADPH-menadione oxidoreductase (NMOR) activity were unaffected by thyroid status, whereas 7-ethoxycoumarin O-deethylase (ECOD) activity was approx. 50% lower in ThX animals than in ad libitum or pair-fed controls. At 3 and 10 days after TCDD administration (10 micrograms/kg, i.p.), P-450 concentrations and NMOR and ECOD activities were induced by approximately the same proportions in ThX and pair-fed intact rats; however, the absolute levels of the induced activities were lower in ThX than in pair-fed controls. It was concluded that hypothyroidism does not regulate Ah receptor concentration or function in the liver. Therefore, the modulation of TCDD toxicity by hypothyroidism appears not to involve changes in the hepatic Ah receptor.     

Restorative effect of various androgens on copulatory behavior of the male golden hamster

Adult male hamsters were castrated, and four weeks later, after a single mating test, began receiving daily 100 μg injections of testosterone (T), androstenedione (AD), or testosterone propionate (TP). Mating tests were conducted at regular intervals during the eight week treatment period. Results indicated the superiority of TP in reinstating intromissions and ejaculations. The T and AD groups did not differ on any of the measures, a result which agrees with previous similar experiments using rats.     

Androgen receptor immunoreactivity in the male and female Syrian hamster brain.

To investigate potential mechanisms for sex differences in the physiologic response to androgens, the present study compared the hormonal regulation of intracellular androgen receptor partitioning and the distribution of androgen receptor immunoreactivity in select brain regions from male and female hamsters. Androgen receptors were visualized on coronal brain sections. Two weeks after castration, androgen receptor immunoreactivity filled the neuronal nuclei and cytoplasm in males and females. In gonad-intact males and females, androgen receptor immunoreactivity was limited to the cell nucleus. Whereas exogenous dihydrotestosterone prevented cytoplasmic immunoreactivity, estrogen at physiologic levels did not. These results suggest that nuclear androgen receptor immunoreactivity in gonad-intact females is maintained by endogenous androgens, and that androgens have the potential to influence neuronal activity in either sex. However, sex differences in the number and staining intensity of androgen-responsive neurons were apparent in select brain regions. In the ventral premammillary nucleus, ventromedial nucleus of the hypothalamus, and medial amygdaloid nucleus, androgen receptor staining was similar in gonadectomized males and females. In the lateral septum, posteromedial bed nucleus of the stria terminalis (BNSTpm), and medial preoptic nucleus, the number of androgen receptor-immunoreactive neurons was significantly lower in females (p < .05). Moreover, the integrated optical density/cell in BNSTpm was significantly less in females (1.28+/-0.3 units) than in males (2.21+/-0.2 units; p < .05). These sex differences in the number and staining intensity of androgen-responsive neurons may contribute to sex differences in the behavioral and neuroendocrine responses to androgens.     

Predictors of dominance in the male golden hamster (Mesocricetus auratus)

The outcome of social interactions between four male hamsters was significantly related to body weight (r_s=0·66) and to the size and pigmentation of their lateral flank glands (r_s=0·82). Weight was held constant in a second experiment and variations in gland size and pigmentation remained significantly related to the outcome of social encounters (r_s=0·77). In a third experiment, castrate hamsters of uniform weight receiving either no hormone, 0·1 mg, 0·50 mg, or 1·00 mg testosterone propionate exhibited flank gland variations significantly related to social rank (r_s=0·81). These experiments demonstrate that the state of the flank gland, which is related to endogenous androgen levels, can be used as a predictor of social rank in male hamsters.     

Effects of cyclophosphamide on the secondary palate development in golden Syrian hamster: teratology, morphology, and morphometry

Cyclophosphamide (CP), when injected in hamster mother between days 9 and 11 of pregnancy, was teratogenic in fetuses. On the basis of a morphological study it was deduced that CP delayed the reorientation of hamster palatal shelves by 16-20 h. In a subsequent experiment, in both control and CP-treated palatal shelves, the numbers of epithelial and mesenchymal cells were counted and cross-sectional area was measured. DNA synthesis, measured by 3H-thymidine incorporation, was used as an index of growth by cell proliferation. The results showed that during the vertical development of palatal shelves, the mesenchymal cells reached their peak number during the initial 24 hours, i.e., at the end of the second peak in DNA synthesis, and remained unchanged thereafter throughout reorientation. The shelf area also showed rapid increase during the initial 24 h followed by a spurt 2 h prior to reorientation. Cyclophosphamide prolonged the acquisition of these features by affecting the mesenchymal cells and consequently delayed the reorientation of the vertical shelves until such time that the number of healthy mesenchymal cells and shelf area were restored to the control values. The data lend further support to the hypothesis that the acquisition of a specific number of cells and shelf volume, during vertical palatal development, may be essential for palatal shelf reorientation.     

Induction of P-450 isoenzyme activities in Syrian golden hamster liver compared to rat liver as probed by the rate of 7-alkoxyresorufin-O-dealkylation

The activities of 7-ethoxyresorufin-O-deethylase (EROD), 7-pentoxyresorufin-O-deethylase (PROD), 7-ethoxycoumarin-O-deethylase (ECOD) and aromatic hydrocarbon hydroxylase (AHH) were measured in hepatic microsomes from male and female Wistar rats and Syrian golden hamsters in order to probe the basal activity and the inducibility by phenobarbital (PB) and 3-methylcholanthrene (MC) of different P-450 isoenzymes. The basal activities of EROD and ECOD, but not PROD and AHH, were higher in male hamsters than in male rats. No sex-related difference in enzyme activities was observed with hamsters, whereas male rats had a higher ECOD and AHH activity than female rats. Induction by PB led to a 450-fold and 250-fold increase in PROD activity in male and female rat liver microsomes, respectively, while MC had a more pronounced inductive effect on EROD activity in this species. In hamsters, EROD activity was induced by MC but not by PB. Unexpectedly PROD activity in male and female hamster liver microsomes was only moderately induced by PB, the extent being lower than on induction by MC. Therefore, the activity of PROD, which is useful as a specific enzymatic assay for P-450 IIB in the rat liver, cannot be used to probe PB-like inducers in the hamster liver.     

Photoperiodic control of meiosis in the male Syrian hamster (Mesocricetus auratus)

Male Syrian hamsters exposed to short photoperiods of 6 h light/day (6L:18D) show regression of the testes within 12 weeks. Chromosome preparations of the meiotic stages (pachytene, metaphase I (MI) and metaphase II (MII)), testicular weights relative to body weights, sperm counts, seminiferous tubule diameter and histological appearance were examined at intervals during regression and subsequent recovery in a long photoperiod (14L:10D). The fall of testicular weight was associated with the decrease in tubule diameter. Spermatogenesis and sperm count were reduced rapidly and finally ceased after 10 weeks in short days. The numbers of MI and MII cells relative to 100 pachytene cells progressively decreased during the short-day treatment, although the ratio of MI:MII stayed constant whenever there was meiotic activity (except in the first week of the recovery phase). This suggests that an increasing proportion of pachytene cells did not progress to MI with increased time in short days, but cells which did reach MI progressed to MII in the same proportions as in the control testes. Meiosis ceased after 10 weeks in short days. Recovery in the long days was marked by a peak in the number of MI and MII cells/100 pachytene cells soon after the return to long days. This preceded the return (to control values) of the sperm count by 10 weeks. Initial recovery in the first 3 weeks was very rapid in all the determined values.     

Effects of hypomagnetic field on noradrenergic activities in the brainstem of golden hamster

Previous studies found that elimination of the geomagnetic field (GMF) interferes with the normal brain functions, but the underlying mechanism remains unknown. The present study examined the effects of long-term exposures to a near-zero magnetic environment on the noradrenergic activities in the brainstem of golden hamsters. Both the content of norepinephrine (NE) and the density of NE-immunopositive neurons in the tissue decreased significantly after the treatment, and the effects could be progressive with time. These variations may substantially contribute to behavioral and mood disorders reported in other studies when animals are shielded from the GMF.     

Effects of dietary selenium on lipid peroxidation, mitochondrial function and protein profiles in the heart of the myopathic Syrian golden hamster (BIO 14.6)

Male, weanling myopathic Syrian Golden Hamsters (BIO 14.6 strain) were fed a selenium-adequate diet (controls) or this diet supplemented with 1.0 ppm selenium (treated) for 30 days. Se-treated animals exhibited a 50% reduction in lipid peroxidation in heart homogenates relative to controls and a 61% increase in mitochondrial glycerophosphate acyltransferase activity. Gel electrophoresis revealed no alterations in cardiac protein profiles from treated or control animals. These data suggest that the selenium prevents peroxidative injury and maintains mitochondrial function in the absence of alterations in cardiac proteins.     

The effects of diethylnitrosamine on ribonucleic acid and protein synthesis in the liver and lung of the Syrian golden hamster

1. Syrian golden hamsters were treated with a single subcutaneous dose of 200mg of diethylnitrosamine/kg. In the liver the treatment produced a significant and early inhibition of the incorporation of orotic acid into RNA and of leucine into protein. Diethylnitrosamine also lowered basal and 20-methylcholanthrene-stimulated activities of hepatic aryl hydrocarbon hydroxylase. 2. RNA synthesis, protein synthesis and aryl hydrocarbon hydroxylase activity were also evaluated in the lungs of the same animals. In this organ only protein synthesis was affected by diethylnitrosamine, but not RNA synthesis or aryl hydrocarbon hydroxylase activity. 3. The incorporation of thymidine into DNA was inhibited in both organs early after diethylnitrosamine treatment and increased 2–3 days later. 4. Although diethylnitrosamine, injected subcutaneously, accumulates in liver and lung in toxicologically active amounts, the acute biochemical responses of the two organs are not identical.