Phytoestrogens alter the reproductive organ development in the mink (Mustela vison)

The aim of the present study was to examine the reproductive effects of two perorally applied phytoestrogens, genistein (8 mg/kg/day) and beta-sitosterol (50 mg/kg/day), on the mink (Mustela vison) at human dietary exposure levels. Parental generations were exposed over 9 months to these phytoestrogens and their offspring were exposed via gestation and lactation. Parents and their offspring were sampled 21 days after the birth of the kits. Sex hormone levels, sperm quality, organ weights, and development of the kits were examined. The exposed females were heavier than the control females at the 1st postnatal day (PND). The control kits were heavier than the exposed kits from the 1st to the 21st PND. Phytoestrogens did not affect the organ weights of the adult minks, but the relative testicular weight of the exposed kits was higher than in the control kits. The relative prostate weight was higher and the relative uterine weight lower in the beta-sitosterol-exposed kits than in the control kits. Moreover, the plasma dihydrotestosterone levels were lower in the genistein-exposed male kits compared to the control male kits. This study could not explain the mechanisms behind these alterations. The results indicate that perinatal phytoestrogen exposures cause alterations in the weight of the reproductive organs of the mink kits.     

Pharmacological profiles of β-funaltrexamine (β-FNA) and β-chlornaltrexamine (β-CNA) on the mouse vas deferens preparation

The profiles of action of β-funaltrexamine (β-FNA) and β-chlornaltrexamine (β-CNA) have been assessed in the mouse vas deferens preparation. β-FNA, but not β-CNA, demonstrated a reversible agonist action that appeared to be mediated via κ-receptor interaction. β-CNA produced an irreversible antagonism of μ-, κ- and δ-mediated agonist actions, whereas β-FNA irreversibly antagonized μ-mediated agonist effects only. This selective action of β-FNA could also be seen following administration in vivo. β-CNA and particularly β-FNA should prove valuable in the elucidation of multiple opioid receptors.     

曼性低浓度吸入七氟醚对雌性小鼠受孕及胚胎的影响

目的探讨慢性低浓度吸入七氟醚对雌性小鼠受孕及胚胎的影响。方法选取6周龄雌性昆明小鼠60只,随机分为四组,每组各15只,对照组和七氟醚组,其中,七氟醚按吸入浓度分为30mg／L组、100mg／L组和300mg／L组;七氟醚组每天吸入对应浓度七氟醚6h,连续吸入4周,对照组吸入等量空气。然后将各组小鼠交配受孕后吸入同样浓度的七氟醚和空气至受孕19d时处死,检查各组受孕率、胚胎数及体重发育情况。结果300mg／L组受孕率低于对照组（P〈0．05）,各组畸形率、胎鼠数量差异无统计学意义（P〉O．05）,300mg／L组胎鼠平均体重低于对照组（P〈0．05）。结论慢性低浓度吸入〈100mg／L七氟醚对雌性小鼠受孕无影响．但当浓度〉300mg／L时可能对胎鼠生长有影响。     

Estrogenic effects of 17 beta-aminoestrogens assessed in uteri of rats and mice

Administration of exogenous estrogens has been associated with an increase of thromboembolic events. The 17β-aminoestrogens produce anticoagulant effects contrasting with the procoagulant effects of the natural occurring estradiol in rodents. This work compares the estrogenic effects induced by 17β-aminoestrogens prolame, butolame, pentolame, and estradiol in vivo models. Dose–response curves were performed using immature CD1 mice and Wistar rats. The animals were injected with estradiol or 17β-aminoestrogens (0.01 to 1000 μg/kg), or vehicle. The uterine wet and dry weights were determined. The 17β-aminoestrogens increased uterine weight in a dose-dependent manner. The uterotrophic effect produced by estradiol induced lower ED₅₀ (6.5 and 4 μg/kg) and higher E_max values (+523–350%) in mice as compared with those from the rat, indicating more susceptibility of the mice model. The 17β-aminoestrogens are partial estrogenic agonists with a relative uterotrophic effect of estradiol (100%) from 9–86%. Only the ED₅₀ values of 17β-aminoestrogens in CD1 mice showed a direct correlation to the length of the amine group substitution in C-17 since their efficacy and potency were in the order: prolame>butolame>pentolame.     

Contrasting effects of estradiol and 17 beta-aminoestrogens on blood clotting time in rats and mice

Estrogens have been associated with thromboembolic events. Our group has described the anticoagulant effect of 17β-aminoestrogens in rodents, potentially new alternative estrogenic agents without thrombogenic risk. This work compares the contrasting effects of estradiol and the 17β-aminoestrogens (prolame, butolame, and pentolame) on blood clotting time. Ovariectomized CD1 mice received a single injection of 17β-aminoestrogens, estradiol (20 to 80 mg/kg), or vehicle. Estradiol decreased blood clotting time from −10% to −25% (48 h; P<0.01) and 17β-aminoestrogens increased it, differing in latency (12 h; +48%, P<0.01) and duration (72 h +58%, P<0.01). In male Wistar rats, similar effects (pentolame +45%; estradiol −31%; P<0.01) were observed 48 h after five consecutive daily injections of 1000 μg/animal/day. The maximum procoagulant effect of estradiol was obtained after 72 h with 10 μg/animal/day (−45%; P<0.01). 17β-Aminoestrogens always produced opposite effects to those of estradiol on blood coagulation.     

Effect of biogenic amines and gaba on ATPase activities in mouse tissue

ATPase activities were studied in brain, kidney and liver (13,000 x g pellet) fraction from the mouse. Dopamine and norepinephrine added in vitro showed a dose-dependent stimulation of Na⁺ and K⁺ activated and oligomycin sensitive Mg²⁺ ATPase activities in brain but not in kidney and liver tissue fractions. GAB and serotonin had no effect on ATPase in brain, but inhibited oligomycin sensitive Mg²⁺ ATPase activities in kidney and liver. The relationship between the enhancement of ATPase activity by neurotransmitters in brain and the neuronal excitation is discussed.     

Effect of atenolol on cadmium-induced testicular toxicity in male rats

Cadmium-induced stress adversely affects testicular activity and causes sympathetic stimulation. To investigate the effect of atenolol, a β-adrenergic receptor blocker, on testicular androgen synthesis after cadmium treatment, adult Sprague-Dawley strain male rats were given a single sc dose of cadmium chloride 0.45 mg/kg BW. Animals were killed on day 3 after treatment. Adrenal weight, adrenal Δ⁵-3β-hydroxysteroid dehydrogenase (Δ⁵-3β-HSD) activity, serum corticosterone, and brain noradrenaline were increased significantly while testicular Δ⁵-3β-HSD and 17β-HSD activities, serum testosterone, and accessory sex organs weight were decreased. Oral coadministration of atenolol at a dose of 2.0 mg/kg body weight for 3 days resulted in complete protection of adrenal Δ⁵-3β-HSD, testicular Δ⁵-3β-HSD, and 17β-HSD activities, adrenal weight, serum corticosterone, and serum testosterone when compared with cadmium-only group and there were no significant differences in these parameters from the vehicle control values. Simultaneous administration of cadmium and atenolol also protected brain noradrenaline content and reduced the rise of testicular cadmium concentration. All the parameters were similar to control levels in rats treated with atenolol alone. We conclude that atenolol may protect testicular androgen synthesis by inhibiting the action of noradrenaline on testicular Leydig cells and adrenocortical hyperactivity in cadmium-treated rats.     

Alpha1A-adrenoceptors predominate in the control of blood pressure in mouse mesenteric vascular bed

1 The pressor action of the alpha1A-adrenoceptor agonist, A61603 (N-[5-(4,5-dihydro-1H-imidazol-2-yl)-2-hydroxy-5,6,7,8-tetrahydronaphthalen-1-yl] methanesulfonamide) or the alpha1-adrenoceptor agonist phenylephrine, and their blockade by selective alpha1-adrenoceptor antagonists in the mouse isolated mesenteric vascular bed were evaluated. 2 A61603 showed a approximately 235-fold higher potency in elevating perfusion pressure in mesenteric bed compared to phenylephrine. 3 The alpha1A-adrenoceptor selective antagonist RS 100329 (5-methyl-3-[3-[4-[2-(2,2,2,-trifluoroethoxy) phenyl]-1-piperazinyl] propyl]-2,4-(1H)-pyrimidinedione), displaced with high affinity agonist concentration-response curves to the right in a concentration-dependent manner. 4 The alpha1D-adrenoceptor selective antagonist BMY 7378 (8-[2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl]-8-azaspiro[4.5] decane-7,9-dione), did not displace A61603 nor did it block the phenylephrine-induced pressor response. 5 The alpha1B/D-adrenoceptor alkylating antagonist chloroethylclonidine (CEC), caused a rightward shift of the phenylephrine concentration-response curve and reduced its maximum response; however, CEC only slightly modified A61603 evoked contraction. 6 The results indicate that the isolated mouse mesenteric vascular bed expresses alpha1A-adrenoceptors and suggest a very discrete role for 1B-adrenoceptors.     

Maternal exposure to fenarimol promotes reproductive performance in mouse offspring

Although fenarimol is a widely used chlorinated fungicide applied to fruits and vegetables and is a suspected endocrine disrupter, transgenerational studies of low doses of fenarimol exposure are not currently available. The aims of this study are to address the effect of maternal exposure to low doses of fenarimol on the reproductive performance of offspring and to investigate the expression changes of genes associated with this effect. Pregnant mice were orally exposed to low doses (0, 2, 20, and 200 μg/kg body weight) of fenarimol during gestational and lactational periods, and their offspring were assessed. The body and organ weights and anogenital distance (AGD) of mice offspring (F1) maternally exposed to fenarimol were determined, and the reproductive performances of these mice were assessed by mating and ovarian follicular and sperm analyses. Fenarimol-exposed F1 mice had shortened AGDs, increased body weight with altered organ weights, increased number of pups, increased number of ovarian follicles, and enhanced sperm quality. Microarray data showed 82 upregulated and 743 downregulated genes in the ovaries of fenarimol-exposed mice, in which Cyp17a1, Cyp19a1, and ERβ were upregulated. In addition, Nobox, a pivotal gene required for proper folliculogenesis, was significantly increased in the ovaries of F1 mice. In conclusion, maternal exposure to fenarimol promotes normal reproductive function in female mouse offspring by increasing the expression levels of genes crucial for ovarian folliculogenesis, identifying fenarimol as a chemical that stimulates reproductive performance. Thus, consumption of fenarimol-contaminated diets by mothers may possibly alter normal reproductive function of offspring in humans and wildlife.     

Pulmonary responses of acute exposure to ultrafine iron particles in healthy adult rats

As critical constituents of ambient particulate matter, transition metals such as iron may play an important role in health outcomes associated with air pollution. The purpose of this study was to determine the respiratory effects of inhaled ultrafine iron particles in rats. Sprague Dawley rats 10-12 weeks of age were exposed by inhalation to iron particles (57 and 90 microg/m(3), respectively) or filtered air (FA) for 6 h/day for 3 days. The median diameter of particles generated was 72 nm. Exposure to iron particles at a concentration of 90 microg/m(3) resulted in a significant decrease in total antioxidant power along with a significant induction in ferritin expression, GST activity, and IL-1beta levels in lungs compared with lungs of the FA control or of animals exposed to iron particles at 57 microg/m(3). NFkappaB-DNA binding activity was elevated 1.3-fold compared with that of control animals following exposure to 90 microg/m(3) of iron, but this change was not statistically significant. We concluded that inhalation of iron particles leads to oxidative stress associated with a proinflammatory response in a dose-dependent manner. The activation of NFkappaB may be involved in iron-induced respiratory responses, but further studies are merited.     

Bactericidal activity of oral beta-lactam antibiotics in plasma and urine versus isogenic Escherichia coli strains producing broad- and extended-spectrum beta-lactamases

Bacteria harbouring extended-spectrum β-lactamases (ESBLs), derived by mutation from TEM-1, TEM-2 or SHV-1 β-lactamases, have been described world-wide. The in vitro activities of these enzymes against β-lactam antibiotics, including oral cephalosporins, are well recognised. The aim of this investigation was to assess the bactericidal activity of oral β-lactam antibiotics available in Croatia (amoxicillin/clavulanate, cephalexin, cefuroxime, cefadroxil and ceftibuten), in biological fluids against isogenic Escherichia coli strains producing broad-spectrum (TEM-1, TEM-2 and SHV-1) and extended-spectrum β-lactamases (SHV-2, SHV-3, SHV-4, SHV-5, SHV-12). Bactericidal activity of oral β-lactams in plasma and urine was tested in time-kill experiments and by determining bactericidal titres at different time intervals post-dose. The killing rate of antibiotics in urine was slower than in plasma, but faster than in Mueller–Hinton broth. High bactericidal titres in urine were only maintained throughout the whole dosing interval by ceftibuten against strains producing broad-, SHV-2 and SHV-3 β-lactamases. The older generation cephalosporins can be considered for the therapy of urinary tract infections caused by E. coli harbouring TEM-1, TEM-2 and SHV-1 β-lactamases but a shorter dosing interval is needed. Ceftibuten can be recommended with caution in ESBL producing E. coli except those producing SHV-4, SHV-5 and SHV-12 that confer resistance to it. If these enzymes are produced, fluoroquinolones or carbapenems could be considered.     

The development of forestomach tumours in the mouse following exposure to 2-butoxyethanol by inhalation: studies on the mode of action and relevance to humans

2-Butoxyethanol, a forestomach carcinogen in mice exposed by inhalation, has been shown to enter the forestomach as a result of grooming and ingestion of material condensed on the skin and fur during exposure. The material entering the stomach concentrates in the forestomach region and persists for at least 48 h post-exposure. Mice given single oral doses of either 2-butoxyethanol or 2-butoxyacetic acid, daily for 10 days, developed a marked hyperkeratosis in the forestomach. 2-Butoxyacetic acid was more potent than 2-butoxyethanol, the NOEL for the former being 50 mg/kg and for the latter, 150 mg/kg. Although a dose dependent increase in cell replication was also seen with both chemicals, the results were confounded by a high labelling rate in the controls. There was no evidence of significant binding of radiolabelled 2-butoxyethanol to proteins in stomach tissues. 2-Butoxyethanol was metabolised in vitro in both mouse and rat forestomach and glandular stomach fractions by alcohol dehydrogenases forming 2-butoxyacetaldehyde which was rapidly converted by aldehyde dehydrogenases to 2-butoxyacetic acid. There was a marked species difference in alcohol dehydrogenase activity between rats and mice with the maximum rates up to one order of magnitude greater in mouse than rat. The alcohol and aldehyde dehydrogenases were heavily concentrated in the stratified squamous epithelium of the forestomach of both rats and mice whereas in the glandular stomach the distribution was more diffuse. In human stomach both enzymes were evenly distributed throughout the epithelial cells of the mucosa. It is concluded that 2-butoxyethanol is ingested following inhalation exposure and concentrates in the forestomach where it is metabolised to 2-butoxyacetic acid which causes cellular damage, increased cell replication and hyperkeratosis. These changes are believed to lead to the tumours seen in mice exposed to 2-butoxyethanol for a lifetime. Differences in structure and enzyme distribution between the rodent and human stomach suggest that the responses seen in the mouse are unlikely to occur in humans.     

Multigenerational reproductive assessment of 4-methylimidazole administered in the diet to Hsd:Sprague Dawley SD rats

The general population, including children and adolescents, is exposed to 4-methylimidazole (4-MI) in the diet. 4-MI is a by-product of caramel color manufacturing. It has been previously classified as a possible human carcinogen and displays potential reproductive toxicity. A follow up assessment of reproductive toxicity was conducted in rats utilizing the reproductive assessment by continuous breeding paradigm, in which multiple generations were exposed to 4-MI in diet at 750, 2500, and 5000 ppm. 4-MI exposure was associated with delays in preputial separation and vaginal opening, impairment in reproductive performance, and concomitant histopathological findings in the prostate, testis, and epididymis at 2500 and 5000 ppm. The Lowest Observed Adverse Effect Level for reproductive (based on prostate atrophy) and developmental toxicity (based on delays in preputial separation and vaginal opening) was 750 ppm, equivalent to approximately 50−60 mg/kg bw/day.     

Induction of apoptosis by 25-hydroxycholesterol in adult rat Leydig cells: protective effect of 17beta-estradiol

Testicular macrophages can convert cholesterol into 25-hydroxycholesterol which strongly stimulates Leydig cell testosterone production. We demonstrated that 25-hydroxycholesterol reduced cholesterol biosynthesis in adult rat Leydig cells. This oxysterol can also be cytotoxic. As hydroxylated cholesterol can induce apoptosis in various cells, we investigated cell death produced by 25-hydroxycholesterol. Apoptosis was characterized by TUNEL assay and by DAPI test. Addition of 25-hydroxycholesterol, during 24 h, induced a dose dependent increase of apoptosis. This effect was reduced by a treatment with a caspase-3 inhibitor (Ac-DEVD-CHO). 25-Hydroxycholesterol is known to stimulate testosterone production, but an increase of intracellular or culture medium testosterone level does not modify significantly the percentage of apoptotic cells. In contrast, addition of 17β-estradiol (2 nM) induced a decrease of apoptotic cells. These data suggested that this oxysterol can be used by rat Leydig cells in culture for sterol metabolism, but also induces apoptosis which could be inhibited by 17β-estradiol.     

Teratogenic effects in mouse fetuses subjected to the concurrent in utero exposure to miconazole and metronidazole

The potential interactivity of the antimicrobials miconazole and metronidazole in the induction of teratogenic effects was investigated in the present study. Drugs were injected at 60 mg/kg either individually or in combination to pregnant mice on gestation day (GD) 8, 9, or 10. Teratological assessments were carried out on GD 18. A potent teratogenic interaction resulted from miconazole–metronidazole co-administration, causing an increment in axial skeletal defects incidence. While, the individual exposure to miconazole or metronidazole produced axial skeletal defects at frequencies that did not exceed 5.6% in the various treatment groups, the percentage of fetuses with malformed skeleton reached 26% after co-exposure on GD 8 or GD 9. No significant synergism was noted when drugs were co-administered on GD 10. This study shows that a teratogenic interaction can result from miconazole–metronidazole concomitant exposure.     

Kappa opioid receptor agonists inhibit the pilocarpine-induced seizures and toxicity in the mouse

Involvement of the κ opioid receptor in regulation of the pilocarpine-induced seizures and neurodegeneration was studied in mice. Administration of pilocarpine (400 mg/kg i.p.) resulted in a sequence of behavioral alterations including motor limbic seizures. Pretreatment of mice with the selective κ opioid receptor agonist U69,593 (2 and 20 mg/kg i.p.) or PD117,302 (0.1 and 1 mg/kg i.p.) increased the latency of motor seizures and decreased the seizure severity and mortality. Those effects were abolished in animals pretreated with the specific κ opioid receptor antagonist nor-binaltorphimine (Nor-BNI, 10 mg/kg i.p.). Examination of frontal forebrain sections by light microscopy revealed widespread damage, especially within the hippocampal formation, in pilocarpine-treated mice. Both U69,593 and PD117,503 protected the integrity of hippocampal neurons, especially in the CA1 region, that effect being reveresed by Nor-BNI. The above data indicate that activation of the κ opioid receptor exerts an inhibitory effect on the pilocarpine-induced limbic seizures and neurotoxicity.     

The role of beta-lactamase producing bacteria and bacterial interference in streptococcal tonsillitis

The causes of penicillin failure in eradicating group A β-haemolytic streptococcal pharyngo-tonsillitis are described. The mechanisms accounting for the failure include the presence in the tonsils of β-lactamase producing bacteria and the absence of bacterial interference. The use of antimicrobials that can overcome and modulate these two phenomena and achieve better cure of the infection is described.