Influence of gender and stress on the volatile sulfur compounds and stress biomarkers production.

BACKGROUND: Stress and menstrual cycle have been described as factors influencing bad breath, as they can alter oral homeostasis and contribute to the production of volatile sulfur compounds (VSC). OBJECTIVE: Considering that the experimenter's and volunteer's gender may influence the volunteer's responses to stress, the aim of this work was to evaluate the influence of stress and gender on the production of VSC and salivary biomarkers. METHODS: The experimental acute stress was induced by the Video-Recorded Stroop Color-Word Test (VRSCWT). The VSC, salivary proteins, and cardiovascular parameters were measured before and after VRSCWT. RESULTS: The VRSCWT induced significant increase in total VSC, hydrogen sulfide, and blood pressure values in men and women. Women presented higher values of both these compounds than men. The increase in systolic blood pressure was more pronounced when subjects were evaluated by an experimenter of the opposite gender. When women were evaluated by a member of the opposite gender, they showed significant increases in salivary alpha-amylase and cortisol compared with baseline values. CONCLUSION: Thus, the results showed that VRSCWT induced acute stress, which increased VSC production, and these effects were shown to be influenced by the gender.

Chronic stress, but not hypercaloric diet, impairs vascular function in rats.

The aim of this study was to evaluate vascular and metabolic effects of chronic mild unpredictable stress (CMS) and hypercaloric diet (HD) without carbohydrate supplementation in rats. Male Sprague-Dawley rats were randomly assigned to four groups: Control, HD, CMS, and HD plus CMS. CMS consisted of the application of different stressors for 3 weeks. The rats were killed 15 days after CMS exposure. The HD group presented higher plasma lipid concentrations, without changes in fasting glucose concentration, glucose tolerance test, and vascular function and morphology, in comparison with the control group. Stressed rats presented higher fasting blood concentration of insulin, higher homeostasis model assessment index values and area under the curve in an oral glucose tolerance test, in comparison with non-stressed rats. CMS increased the plasma concentrations of corticosterone and lipids, and the atherogenic index values, without change in high-density lipoprotein level. CMS increased intima-media thickness and induced endothelium-dependent supersensitivity to phenylephrine, and lowered the relaxation response to acetylcholine in the thoracic aorta isolated from rats fed with control or HD, in comparison with non-stressed groups. CMS effects were independent of diet. In non-stressed rats, the HD induced dyslipidemia, but did not change glucose metabolism, vascular function, or morphology. The data from this study indicate that CMS promotes a set of events which together can contribute to impair function of the thoracic aorta.

Proatherosclerotic effects of chronic stress in male rats: altered phenylephrine sensitivity and nitric oxide synthase activity of aorta and circulating lipids.

The aim of this study was to analyze the effects of chronic mild unpredictable stress (CMS) on the vasoconstrictor response and morphology of the thoracic aorta and serum lipid profiles in rats. Male Sprague-Dawley rats were submitted to CMS, which consisted of the application of different stressors for 7 days per week across 3 weeks. The rats were sacrificed 15 days after CMS exposure. CMS induced supersensitivity to the vasoconstrictor effect of phenylephrine in endothelium-intact thoracic aortic rings without changes in aortic rings without endothelium, or pre-incubated with nitric oxide (NO) synthesis inhibitor. Rats submitted to CMS showed hypertrophy of the intima and tunica media of thoracic aorta, increased serum levels of triglycerides, total cholesterol, very low-density lipoprotein cholesterol, low-density lipoprotein cholesterol and atherogenic index, without changes in high-density lipoprotein cholesterol levels, when compared with control rats. These data indicate that CMS induces physiological and morphological changes that may contribute to the development of atherosclerosis by mechanisms related to deficiency in NO production and dyslipidemia.

Influence of estradiol and progesterone on the sensitivity of rat thoracic aorta to noradrenaline.

The aim of this study was to investigate the effects of low and high doses of estradiol, and of progesterone on the response to noradrenaline in rat thoracic aorta. Two weeks after bilateral ovariectomy, female rats received a s.c. injection of vehicle (corn oil, 0.1 mL/day), estradiol (10 microg/kg/day or 4 mg/kg/day) and/or progesterone (20 mg/kg/day), for eight days. On the ninth day, the rats were sacrificed and aortic rings, with or without endothelium, were used to generate concentration-response curves to noradrenaline. Aortic rings with intact endothelium from the high-dose (4 mg/kg/day) estradiol group were supersensitive to noradrenaline compared to the vehicle or low-dose (10 microg/kg/day) estradiol groups (pD2 values = 7.86+/-0.09, 7.30+/-0.11 and 7.35+/-0.04, respectively). Endothelium-intact aortic rings from high-estradiol rats were supersensitive to noradrenaline when compared to vehicle-, progesterone- and progesterone + high-estradiol-treated rats (pD2 values = 7.77+/-0.12, 7.21+/-0.13, 6.93+/-0.04 and 7.22+/-0.18, respectively). There were no significant differences among the pD2 values for noradrenaline in aortic rings without endothelium. In conclusion, at high but not low doses, estradiol increased the sensitivity to noradrenaline and this was prevented by progesterone. Both of these effects were endothelium-dependent.

Estrous cycle influences the response of female rats in the elevated plus-maze test.

The aim of this study was to examine the state of anxiety and the 17beta-estradiol and progesterone levels in rats tested in the elevated plus-maze during the four phases of the estrous cycle. Male rats, female rats during each of the four phases of the estrous cycle, ovariectomized rats, and diestrus female rats treated with estradiol were tested in the elevated plus-maze between 8:00 and 10:00 a.m. Blood was collected from all rats for the determination of 17beta-estradiol and progesterone levels. Female rats in the proestrus group spent more time in the open arms than diestrus rats (P<.05). There were no significant differences in the percentage of entries into the open arms or in the number of entries into the closed arms among the phases of the estrous cycle or between males and normal or ovariectomized females. Serum estradiol levels were higher (P<.05) during proestrus compared to estrus, metestrus, and diestrus in control and plus-maze tested female rats, but there were no significant differences in progesterone levels. Treating diestrus female rats with estradiol to produce estradiol plasma concentrations similar to those seen during proestrus abolished the difference in the percentage of time spent in the open arms by proestrus and diestrus rats. Since the time spent in the open arms of the plus-maze is inversely related to anxiety, we conclude that the anxiety levels of female rats were lower in proestrus than during diestrus, and that the levels of estradiol modulate this response.

Determination of the estrous cycle phases of rats: some helpful considerations.

The short length of the estrous cycle of rats makes them ideal for investigation of changes occurring during the reproductive cycle. The estrous cycle lasts four days and is characterized as: proestrus, estrus, metestrus and diestrus, which may be determined according to the cell types observed in the vaginal smear. Since the collection of vaginal secretion and the use of stained material generally takes some time, the aim of the present work was to provide researchers with some helpful considerations about the determination of the rat estrous cycle phases in a fast and practical way. Vaginal secretion of thirty female rats was collected every morning during a month and unstained native material was observed using the microscope without the aid of the condenser lens. Using the 10 x objective lens, it was easier to analyze the proportion among the three cellular types, which are present in the vaginal smear. Using the 40 x objective lens, it is easier to recognize each one of these cellular types. The collection of vaginal lavage from the animals, the observation of the material, in the microscope, and the determination of the estrous cycle phase of all the thirty female rats took 15-20 minutes.

Influence of gender and menstrual cycle on volatile sulphur compounds production.

The menstrual cycle has been pointed out as a factor influencing halitosis. However, this relationship has not yet been clarified. The aim of this study was to evaluate the influence of gender and the menstrual cycle on the production of volatile sulphur compounds (VSC) in women (n=14) across the menstrual cycle, and in men (n=17). Volunteers in good oral and general health were submitted to the evaluation of VSC, salivary flow, cortisol and anaerobic bacteria counts in saliva. Data were compared among groups by Analysis of Variance (alpha=5%). VSC was higher in the menstrual and premenstrual phases when compared with men and the follicular phase (p<0.05). Salivary flow was lower in the menstrual and premenstrual phases when compared with men and the follicular phase (p<0.05). Salivary cortisol was higher in the menstrual phase in comparison with men and the premenstrual and follicular phases (p<0.05). Total salivary protein was higher in men when compared to women (p<0.05) with no differences among menstrual phases (p>0.05). Levels of anaerobic micro-organisms, however, were not different among groups (p>0.05). In conclusion, the production of VSC is influenced by menstrual cycle and protein concentration and salivary flow might be involved in this process.

Influence of anabolic steroid on anxiety levels in sedentary male rats.

The aim of this study was to evaluate the influence of nandrolone decanoate on anxiety levels in rats. Male Wistar rats were treated with nandrolone decanoate (5mg/kg, two times per week, i.m.) or vehicle (propylene glycol--0.2 ml/kg, two times per week, IM) for 6 weeks. Control rats were subject only to procedures related to their routine husbandry. By the end of 6 weeks, all groups (24-29 rats/group) were submitted to the elevated plus maze test in order to evaluate their anxiety level. Some of these animals (12-14/group) were treated with diazepam (1 mg/kg i.p.) 30 min before the elevated plus maze test. Nandrolone decanoate significantly decreased the percentage of time spent in the open arms (1.46+/-0.49%) compared with control (3.80+/-0.97%) and vehicle (3.96+/-0.85%) groups, with no difference between control and vehicle treatments. The percentage of open arm entries was also reduced in the group treated with nandrolone decanoate in comparison with the vehicle and control. No changes in the number of closed arm entries were detected. Diazepam abolished the effects of nandrolone decanoate on the percentage of time in, and entries into the open arms. The present study showed that chronic treatment with a high dose of nandrolone decanoate increased the anxiety level in male rats.

Oral concentration of volatile sulphur compounds in stressed rats.

Stress has been identified as a halitosis-inducing factor. Halitosis may be measured by the determination of oral volatile sulphur compound levels (VSC). Since immobilization and swimming are two experimental protocols widely used to induce stress in laboratory animals, the aim of this work was to investigate the influence of stress on VSC in rats. Male Wistar rats were submitted to three swimming or immobilization sessions over consecutive days. The oral VSC increased 3 h after the first and third swimming or immobilization sessions. The results in the present study support the hypothesis that stress may be an etiological factor in halitosis. Also, the animal experimental design may represent a new approach to research concerning the relationship between halitosis and stress.

Relationship among sensitivity to adrenaline, plasma corticosterone level, and estrous cycle in rats.

The dose-response curves to the chronotropic effect of adrenaline obtained in right atria isolated from female rats indicated an order of increasing sensitivity to adrenaline, at the pD2 level, according to the estrous cycle, as follows: estrus < or = metestrus < or = diestrus < or = proestrus. Inhibition of neuronal and extraneuronal uptake shifted the dose-response curves to adrenaline to the left only in right atria isolated from rats during estrus or metestrus. Moreover, under these experimental conditions, right atria were subsensitive to adrenaline during proestrus, in contrast to metestrus. Plasma corticosterone levels were lower during estrus and higher at proestrus. There was a positive correlation between right atria sensitivity to adrenaline and plasma corticosterone levels and estrous cycle phases. Our results also suggest that in the rat atria during proestrus, as opposed to the other phases of the estrous cycle, there was an endogenous inhibition of extraneuronal uptake together with some alteration at the adrenoceptor level and (or) at intracellular mechanisms beyond receptors.

The beta1-adrenoceptor site activated by CGP12177 varies in behavior according to the estrous cycle phase and stress.

The aim of this work was to assess whether stress and estrous cycle phases affected the beta1-adrenoceptor (beta1-AR) site activated by CGP12177 in the right atria of rats. The chronotropic response to CGP12177 in the absence or presence of antagonists was determined in atria from rats submitted to one daily foot-shock session for 3 consecutive days. Blood was collected for hormonal assays. The pD2 for CGP12177 in atria from females was lower than in atria from males and was unaltered by stress or the estrous cycle. Propranolol (200 nM) or CGP20712A (3 microM) shifted the concentration-response curves to CGP12177 to the right in control and stressed estrus or control diestrus rats. Atria from stressed diestrus rats were resistant to blockade by propranolol or CGP20712A, indicating that the effect of beta-adrenoceptor antagonists on the response to CGP12177 is influenced by estrous cycle phases. The stress-induced increase in serum corticosterone levels was independent of the estrous cycle or gender, but the estradiol/progesterone ratio was affected differently in the two groups of female rats. In the diestrus group, serum estradiol levels decreased after the first foot-shock session and remained low until the day of sacrifice, whereas in the estrus group the serum levels of estradiol did not decrease after stress and peaked on the second day, which corresponded to proestrus. These data do not indicate whether there is a direct or indirect effect of stress hormones and (or) sex steroids on cardiac beta1-AR sensitivity. However, they do show that the classic and low-affinity binding sites of the beta1-AR are independently regulated and that the beta1-AR atypical site affinity for antagonists depends on the estrous cycle.

Stress-induced subsensitivity to catecholamines depends on the estrous cycle.

In this article we compare how sensitivity to the chronotropic effect of noradrenaline and adrenaline of right atria isolated from female rats is modified after repeated swimming or foot-shock stress, under the influence of the estrous cycle. Right atria from stressed female rats sacrificed at diestrus were subsensitive to both catecholamines, irrespective of the stressor agent. However, although subsensitivity to noradrenaline was of similar intensity, subsensitivity to adrenaline was more pronounced in right atria from foot shock stressed rats as opposed to swimming-stressed rats. Identical stress protocols did not induce any alteration in atrial sensitivity to catecholamines when the stressed female rats were sacrificed at estrus. We conclude that the stress reaction concerning the mediation of cardiac chronotropism by catecholamines is related to the severity of the stressor agent and is strongly influenced by the estrous cycle.

Influence of the estrous cycle on the sensitivity to catecholamines in right atria from rats submitted to foot-shock stress.

We investigated the mechanisms of the alterations in sensitivity to catecholamines in right atria from female rats exhibiting regular 4-day estrous cycles after three foot-shock sessions at estrus, metestrus, and diestrus or at diestrus, proestrus, and estrus. Right atria from stressed rats sacrificed at diestrus showed subsensitivity to noradrenaline and adrenaline. After in vitro sympathetic denervation (38 microM 6-hydroxydopamine) plus inhibition of neuronal reuptake (0.1 microM desipramine) subsensitivity to noradrenaline was abolished, but it was again evident when extraneuronal uptake was also inhibited (10 microM phenoxybenzamine and 30 microM corticosterone). The same pretreatment abolished the subsensitivity to adrenaline. After addition of 1 microM butoxamine, a beta 2-adrenoceptor antagonist, the tissues from stressed rats were subsensitive to adrenaline. Right atria from stressed rats sacrificed at estrus did not show any alteration in sensitivity to catecholamines. We conclude that after foot-shock stress, right atria from female rats sacrificed at diestrus showed subsensitivity of the chronotropic response to catecholamines as a result of a conformational alteration of beta 1-adrenoceptors, simultaneously with an increase in beta 2-adrenoceptor-mediated response. The mechanisms seem to be similar to those which underlie stress-induced alterations in catecholamine sensitivity in right atria from male rats. However, during estrus there are some protective factors that prevent the effects of stress on right atria.

Pharmacological evidence for beta2-adrenoceptor in right atria from stressed female rats.

The purpose of the present study was to demonstrate a physiological response to TA2005, a potent m-adrenoceptor (beta2-AR) selective agonist, in right atria isolated from stressed female rats under the influence of the estrous cycle. We obtained concentration-response curves to the agonist in the presence and in the absence of selective antagonists in right atria isolated from female rats submitted to three daily foot-shock sessions (30 min duration, 120 pulses of 1.0 mA, 1.0 s, applied at random intervals of 5-25 s) and sacrificed at estrus or diestrus. Our results showed that the pD2 values of TA2005 were not influenced by estrous cycle phase or foot-shock stress. However, in right atria from stressed rats sacrificed during diestrus, the concentration-response curve to TA2005 was biphasic, with a response being obtained at concentrations of 0.1 nM, whereas during estrus no response was observed at doses lower than 3 nM. ICI 1118,551, a beta2-AR antagonist, abolished the response to nanomolar concentrations of TA2005 in right atria from stressed rats at diestrus, with no changes in agonist pD2 values in right atria from control rats (7.47 +/- 0.09, p > 0.05) but a 3-fold decrease in pD2 values of TA2005 in right atria from foot shock stressed rats (7.90 +/- 0.07, p < or = 0.05). Concentration-response curves to TA2005 in the presence of ICI118,551 were best fitted by a one-site model equation. The beta1-AR antagonist, CGP20712A, shifted to the right only the second part of the concentration-response curves to the agonist, unmasking the putative beta2-AR-mediated response to the agonist in tissues isolated from stressed rats at diestrus. Under this condition, concentration-response curves to the agonist were best fitted by a two-site model equation. pD2 and maximum response of TA2005 interaction with beta1- and putative beta2-adrenoceptor components were calculated. Schild analyses gave a pK(B) value for CGP20712A that was typical for the interaction with beta1-AR in each experimental group. pK(B) values for ICI118,551 could not be obtained in stressed rats sacrificed at diestrus since Schild plot slopes were lower than 1.0. In right atria from control rats, ICI118,551 pK(B) values were similar to reported values for the interaction of the antagonist with beta1-AR. These results confirm that a heterogenous beta-AR population mediating the chronotropic response to catecholamines can be demonstrated in right atria from foot shock stressed female rats sacrificed at diestrus. The stress-induced response seems to be mediated by the beta2-AR subtype. Right atria from rats sacrificed during estrus are protected against stress-induced alterations on the homogeneity of beta-AR population.

Determination of the estrous cycle phases of rats: some helpful considerations

The short length of the estrous cycle of rats makes them ideal for investigation of changes occurring during the reproductive cycle. The estrous cycle lasts four days and is characterized as: proestrus, estrus, metestrus and diestrus, which may be determined according to the cell types observed in the vaginal smear. Since the collection of vaginal secretion and the use of stained material generally takes some time, the aim of the present work was to provide researchers with some helpful considerations about the determination of the rat estrous cycle phases in a fast and practical way. Vaginal secretion of thirty female rats was collected every morning during a month and unstained native material was observed using the microscope without the aid of the condenser lens. Using the 10 x objective lens, it was easier to analyze the proportion among the three cellular types, which are present in the vaginal smear. Using the 40 x objective lens, it is easier to recognize each one of these cellular types. The collection of vaginal lavage from the animals, the observation of the material, in the microscope, and the determination of the estrous cycle phase of all the thirty female rats took 15-20 minutes