Developmental and behavioral effects of postnatal amitraz exposure in rats

The effects of postnatal amitraz exposure on physical and behavioral parameters were studied in Wistar rats, whose lactating dams received the pesticide (10 mg/kg) orally on days 1, 4, 7, 10, 13, 16 and 19 of lactation; control dams received distilled water (1 ml/kg) on the same days. A total of 18 different litters (9 of them control and 9 experimental) born after a 21-day gestation were used. The results showed that the median effective time (ET50) for fur development, eye opening, testis descent and onset of the startle response were increased in rats postnatally exposed to amitraz (2.7, 15.1, 21.6 and 15.3 days, respectively) compared to those of the control pups (1.8, 14.0, 19.9 and 12.9 days, respectively). The ages of incisor eruption, total unfolding of the external ears, vaginal and ear opening and the time taken to perform the grasping hindlimb reflex were not affected by amitraz exposure. Pups from dams treated with amitraz during lactation took more time (in seconds) to perform the surface righting reflex on postnatal days (PND) 3 (25.0 ± 2.0), 4 (12.3 ± 1.2) and 5 (8.7 ± 0.9) in relation to controls (10.6 ± 1.2; 4.5 ± 0.6 and 3.4 ± 0.4, respectively); the climbing response was not changed by amitraz. Postnatal amitraz exposure increased spontaneous motor activity of male and female pups in the open-field on PND 16 (140 ± 11) and 17 (124 ± 12), and 16 (104 ± 9), 17 (137 ± 9) and 18 (106 ± 8), respectively. Data on spontaneous motor activity of the control male and female pups were 59 ± 11 and 69 ± 10 for days 16 and 17 and 49 ± 9, 48 ± 7 and 56 ± 7 for days 16, 17 and 18, respectively. Some qualitative differences were also observed in spontaneous motor behavior; thus, raising the head, shoulder and pelvis matured one or two days later in the amitraz-treated offspring. Postnatal amitraz exposure did not change locomotion and rearing frequencies or immobility time in the open-field on PND 30, 60 and 90. The present findings indicate that postnatal exposure to amitraz caused transient developmental and behavioral changes in the exposed offspring and suggest that further investigation of the potential health risk of amitraz exposure to developing human and animal offsprings may be warranted.

Effects of estradiol benzoate on 5'-iodothyronine deiodinase activities in female rat anterior pituitary gland, liver and thyroid gland

There is little information on the possible effects of estrogen on the activity of 5'-deiodinase (5'-ID), an enzyme responsible for the generation of T3, the biologically active thyroid hormone. In the present study, anterior pituitary sonicates or hepatic and thyroid microsomes from ovariectomized (OVX) rats treated or not with estradiol benzoate (EB, 0.7 or 14 µg/100 g body weight, sc, for 10 days) were assayed for type I 5'-ID (5'-ID-I) and type II 5'-ID (5'-ID-II, only in pituitary) activities. The 5'-ID activity was evaluated by the release of 125I from deiodinated 125I rT3, using specific assay conditions for type I or type II. Serum TSH and free T3 and free T4 were measured by radioimmunoassay. OVX alone induced a reduction in pituitary 5'-ID-I (control = 723.7 ± 67.9 vs OVX = 413.9 ± 26.9; P<0.05), while the EB-treated OVX group showed activity similar to that of the normal group. Thyroid 5'-ID-I showed the same pattern of changes, but these changes were not statistically significant. Pituitary and hepatic 5'-ID-II did not show major alterations. The treatment with the higher EB dose (14 µg), contrary to the results obtained with the lower dose, had no effect on the reduced pituitary 5'-ID-I of OVX rats. However, it induced an important increment of 5'-ID-I in the thyroid gland (0.8 times higher than that of the normal group: control = 131.9 ± 23.7 vs ovx + EB 14 µg = 248.0 ± 31.2; P<0.05), which is associated with increased serum TSH (0.6-fold vs OVX, P<0.05) but normal serum free T3 and free T4. The data suggest that estrogen is a physiological stimulator of anterior pituitary 5'-ID-I and a potent stimulator of the thyroid enzyme when employed at high doses

Study of the effects of ß-myrcene on rat fertility and general reproductive performance

ß-Myrcene (MYR) is a monoterpene found in the oils of a variety of aromatic plants including lemongrass, verbena, hop, bay, and others. MYR and essential oils containing this terpenoid compound are used in cosmetics, household products, and as flavoring food additives. This study was undertaken to investigate the effects of MYR on fertility and general reproductive performance in the rat. MYR (0, 100, 300 and 500 mg/kg) in peanut oil was given by gavage to male Wistar rats (15 per dose group) for 91 days prior to mating and during the mating period, as well as to females (45 per dose group) continuously for 21 days before mating, during mating and pregnancy, and throughout the period of lactation up to postnatal day 21. On day 21 of pregnancy one-third of the females of each group were submitted to cesarean section. Resorption, implantation, as well as dead and live fetuses were counted. All fetuses were examined for external malformations, weighed, and cleared and stained with Alizarin Red S for skeleton evaluation. The remaining dams were allowed to give birth to their offspring. The progeny was examined at birth and subsequently up to postnatal day 21. Mortality, weight gain and physical signs of postnatal development were evaluated. Except for an increase in liver and kidney weights, no other sign of toxicity was noted in male and female rats exposed to MYR. MYR did not affect the mating index (proportion of females impregnated by males) or the pregnancy index (ratio of pregnant to sperm-positive females). No sign of maternal toxicity and no increase in externally visible malformations were observed at any dose level. Only at the highest dose tested (500 mg/kg) did MYR induce an increase in the resorption rate and a higher frequency of fetal skeleton anomalies. No adverse effect of MYR on postnatal weight gain was noted but days of appearance of primary coat, incisor eruption and eye opening were slightly delayed in the exposed offspring. On the basis of the data presented in this paper the no-observed-adverse-effect level (NOAEL) for toxic effects on fertility and general reproductive performance can be set at 300 mg of ß-myrcene/kg body weight by the oral route.

Effect of medroxyprogesterone acetate on thyrotropin secretion in adult and old female rats

Steroid hormones have been implicated in the modulation of TSH secretion; however, there are few and controversial data regarding the effect of progesterone (Pg) on TSH secretion. Medroxyprogesterone acetate (MPA) is a synthetic alpha-hydroxyprogesterone analog that has been extensively employed in therapeutics for its Pg-like actions, but that also has some glucocorticoid and androgen activity. Both hormones have been shown to interfere with TSH secretion. The objective of the present study was to investigate the effects of MPA or Pg administration to ovariectomized (OVX) rats on in vivo and in vitro TSH release and pituitary TSH content. The treatment of adult OVX rats with MPA (0.25 mg/100 g body weight, sc, daily for 9 days) induced a significant (P<0.05) increase in the pituitary TSH content, which was not observed when the same treatment was used with a 10 times higher MPA dose or with Pg doses similar to those of MPA. Serum TSH was similar for all groups. MPA administered to OVX rats at the lower dose also had a stimulatory effect on the in vitro basal and TRH-induced TSH release. The in vitro basal and TRH-stimulated TSH release was not significantly affected by Pg treatment. Conversely, MPA had no effect on old OVX rats. However, in these old rats, ovariectomy alone significantly reduced (P<0.05) basal and TRH-stimulated TSH release in vitro, as well as pituitary TSH content. The results suggest that in adult, but not in old OVX rats, MPA but not Pg has a stimulatory effect on TSH stores and on the response to TRH in vitro.

Regulation of antioxidant enzyme activities in male and female rat macrophages by sex steroids

Human and animal immune functions present sex dimorphism that seems to be mainly regulated by sex hormones. In the present study, the activities of the antioxidant enzymes total superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) were measured in intraperitoneal resident macrophages from adult male and female rats. In addition to comparing males and females, we also examined the regulation of these enzyme activities in macrophages by sex steroids. GSH-Px activity did not differ between male and female macrophages. However, both total SOD and CAT activities were markedly higher in females than in males (83 and 180%). Removal of the gonads in both males and females (comparison between castrated groups) increased the difference in SOD activity from 83 to 138% and reduced the difference in CAT activity from 180 to 86%. Castration and testosterone administration did not significantly modify the activities of the antioxidant enzymes in male macrophages. Ovariectomy did not affect SOD or GSH-Px activity but markedly reduced (48%) CAT activity. This latter change was fully reversed by estrogen administration, whereas progesterone had a smaller effect. These results led us to conclude that differences in the SOD and CAT activities may partially explain some of the differences in immune function reported for males and females. Also, estrogen is a potent regulator of CAT in macrophages and therefore this enzyme activity in macrophages may vary considerably during the menstrual cycle.

Induction of liver monooxygenases by annatto and bixin in female rats

Annatto or urucum is an orange-yellow dye obtained from Bixa orellana seeds. It has been used as a natural dye in a variety of food products, drugs and cosmetics, and also in Brazilian cuisine as a condiment ('colorau'). Bixin, a carotenoid devoid of provitamin A activity, is the main pigment found in annatto. Some carotenoids (canthaxanthin, astaxanthin and ß-Apo-8'-carotenal) are known to be potent inducers of CYP1A1, a property not shared by others (ß-carotene, lycopene and lutein). Little is known, however, about the CYP1A1-inducing properties of bixin and annatto. The present study was performed to determine the effects of an annatto extract (28% bixin) and bixin (95% pure) on rat liver monooxygenases. Adult female Wistar rats were treated by gavage with daily doses of annatto (250 mg/kg body weight, which contains approximately 70 mg bixin/kg body weight), bixin (250 mg/kg body weight) or the vehicle only (corn oil, 3.75 g/kg body weight) for 5 consecutive days, or were not treated (untreated control). The activities of aniline-4-hydroxylase (A4H), ethoxycoumarin-O-deethylase (ECOD), ethoxy- (EROD), methoxy- (MROD), pentoxy- (PROD) and benzyloxy- (BROD) resorufin-O-dealkylases were measured in liver microsomes. Annatto (250 mg/kg containing 70 mg bixin/kg) induced EROD (3.8x), MROD (4.2x), BROD (3.3x) and PROD (2.4x). Bixin (250 mg/kg) was a weaker inducer of EROD (2.7x), MROD (2.3x) and BROD (1.9x) and did not alter PROD, A4H or ECOD activities. These results suggest that constituents of the extract other than bixin play an important role in the induction of CYP1A and CYP2B observed with annatto food colorings.

Reproductive ability of pubertal male and female rats

Ten Fisher rats 50 to 55 days of age made up the pubertal group, and ten rats 90 to 95 days of age served as the controls. The testicular and epididymal weights and volumes of the pubertal males were lower than those of the controls (P<0.001). There was also a difference in relative epididymal weight (P<0.001). The sperm of pubertal males was morphologically abnormal in 58.2% of cases, as opposed to only 3.8% in the controls (P<0.001). The mean number of spermatozoa in the control group was 11.9 × 10(6)/ml and their viability was 99.6%, while these values could not be determined for pubertal rats. Serum testosterone was higher in the pubertal animals than in the controls (2.52 ± 1.46 vs 0.92 ± 0.34 nM, P<0.01). The ovaries of control females were heavier than those of pubertal females (P<0.001) but there was no difference in their relative weights. Serum estradiol was similar in both groups (75.5 ± 12.8 vs 81.8 ± 14.7 nM, P>0.05). At the beginning of gestation, the pubertal dams weighed less than the controls (P<0.001) but following uterectomy the body weights were equal. Pubertal dams delivered fewer pups than the controls (8.1 ± 2.5 vs 10.4 ± 1.3, P<0.05). There was no difference in the body weights of their offspring or in the weights of their placentas. The results suggest that, in contrast to their female counterparts, pubertal male rats are not fully mature and have not reached complete reproductive capacity at 50-55 days of age.

Phenotype and genotype correlation of the microconversion from the CYP21A1P to the CYP21A2 gene in congenital adrenal hyperplasia

Deficiency of 21-hydroxylase is the most common form of congenital adrenal hyperplasia (CAH-21OH). We determined by allele-specific PCR the frequency of microconversion in the CYP21A2 gene in 50 Brazilian patients with the classical (salt wasting: SW and simple virilizing: SV) forms and nonclassical (NC) form of CAH-21OH and correlated genotype with phenotype. Genotypes were classified into three mutation groups (A, B, and C) based on the amount of enzymatic activity in in vitro studies using adrenal cells. In 94 unrelated alleles, we diagnosed 76% of the affected alleles after screening for 7 microconversions. The most frequent point mutations observed in this series were I172N (19%), V281L (18%), and IVS2,A/C>G,-12 (15%). In the SW form, the most frequent mutation was IVS2,A/C>G,-12 (38%), in the SV form it was I172N (53%), and in the NC form it was V281L (57.7%). We observed a good correlation between genotype and phenotype. Discordance between genotype and phenotype was found in one SV patient with a mild mutation in one of the alleles (R356W/V281L). However, we cannot rule out the presence of an additional mutation in these alleles. We also observed a good correlation of genotype with 17alpha-hydroxyprogesterone, testosterone, and androstenedione levels. The severity of external genitalia virilization correlated with the severity of mutation. In conclusion, the frequencies described in the present study did not differ from worldwide studies, including the Brazilian population. The few differences observed may reflect individual sample variations. This new Brazilian cohort study suggests the presence of new mutations in Brazilian patients with different forms of CAH-21OH.

The vulnerability of Brazilian female prisoners to HIV infection

The purpose of the present study was to determine the vulnerability of women in prison to HIV infection. The study was carried out from August to October 2000 in a São Paulo State Penitentiary, where 299 female prisoners were serving time. We interviewed and obtained a blood sample from 290 females who agreed to enter the study. Sera were tested for the presence of antibodies to HIV, hepatitis C virus (HCV) and syphilis and the odds ratio (OR) was calculated for variables related to HIV positivity on the basis of a questionnaire. The overall prevalence data were: 13.9% for HIV (37 of 267), 22.8% for syphilis (66 of 290), and 16.2% for HCV (47 of 290). Sexual partnership variables were significantly related to HIV infection. These included HIV-positive partners (OR = 7.36, P = 0.0001), casual partners (OR = 8.96, P = 0.009), injectable drug user partners (OR = 4.7, P = 0.0001), and history of sexually transmitted disease (OR = 2.07, P = 0.05). In addition, a relationship was detected between HIV infection and drug use (OR = 2.48, P = 0.04) and injectable drug use (OR = 4.2, P = 0.002). Even women with only one partner presented a significant OR for HIV infection (OR = 2.57, P = 0.009), reflecting their vulnerability due to their trust in their partner, who did not use a condom. Although the use of injectable substances is associated with HIV infection, our results point to sexual behavior as the most important component of HIV transmission in the female prisoner population.

IgA response in serum and gut secretion in sensitized mice fed with the dust mite Dermatophagoides pteronyssinus extract

Induced oral tolerance to mucosal-exposed antigens in immunized animals is of particular interest for the development of immunotherapeutic approaches to human allergic diseases. This is a unique feature of mucosal surfaces which represent the main contact interface with the external environment. However, the influence of oral tolerance on specific and natural polyreactive IgA antibodies, the major defense mechanism of the mucosa, is unknown. We have shown that oral administration of an extract of the dust mite Dermatophagoides pteronyssinus (Dp) to primed mice caused down-regulation of IgE responses and an increase in tumor growth factor-ß secretion. In the present study, we observed that primed inbred female A/Sn mice (8 to 10 weeks old) fed by gavage a total weight of 1.0-mg Dp extract on the 6th, 7th and 8th days post-immunization presented normal secretion of IL-4 and IL-10 in gut-associated lymphoid tissue and a decreased production of interferon gamma induced by Dp in the draining lymph nodes (13,340 ± 3,519 vs 29,280 ± 2,971 pg/ml). Mice fed the Dp extract also showed higher levels of serum anti-Dp IgA antibodies and an increase of IgA-secreting cells in mesenteric lymph nodes (N = 10), reflecting an increase in total fecal IgA antibodies (N = 10). The levels of secretory anti-Dp IgA antibodies increased after re-immunization regardless of Dp extract feeding. Oral tolerance did not interfere with serum or secretory IgA antibody reactivity related to self and non-self antigens. These results suggest that induction of oral tolerance to a Dp extract in sensitized mice triggered different regulatory mechanisms which inhibited the IgE response and stimulated systemic and secretory IgA responses, preserving the natural polyreactive IgA antibody production.

Female novelty and the courtship behavior of male guinea pigs (Cavia porcellus)

In several rodent species, an increase or recovery of sexual behavior can be observed when sexually satiated males are placed in contact with a novel mate. In order to assess the influence of female novelty on the courtship behavior of guinea pigs (Cavia porcellus), four adult males were observed during four daily 15-min sessions while interacting with the same pregnant female (same-female sessions). A new female was presented during the fifth session (switched-female session). The duration of behavioral categories was obtained from videotape records using an observational software. From the first to the second session, all males decreased the time allocated to investigating (sniffing and licking), following, and mounting the female, and that response did not recover by the end of the same-female sessions. No similar decreasing tendencies were detected in the circling or rumba categories. A marked increase of investigating occurred in all males from the last same-female session (8.1, 11.9, 15.1 and 17.3 percent session time) to the switched-female one (16.4, 18.4, 37.1 and 28.9 percent session time, respectively). Increases in following and circling were recorded in three of four males, and full-blown recovery of mounting in one male. No consistent changes in the females' responses to males (following or attacking) were observed throughout testing. These results are consistent with the hypothesis that guinea pig males recognize individual females and that courtship responses may suffer a habituation/recovery process controlled by mate novelty.

Follicle profile and plasma gonadotropin concentration in pubertal female ponies

Twelve female ponies were examined daily for 30 days and classified as ovulating (OV; N = 6; 197 ± 6 kg) or prepubertal (PP; N = 6; 196 ± 9 kg). Follicles were detected by ultrasound and gonadotropins quantified by radioimmunoassay. The mean diameter of the largest follicles was significantly larger in OV (38 ± 1 mm) than in PP (26 ± 2 mm) but there was no difference between groups in the size of the second largest follicle. There were more small follicles (<24 mm) in the PP than in the OV group, but PP fillies had a smaller number of follicles >29 mm than the OV fillies. Follicle-stimulating hormone (FSH) levels did not differ between groups but PP fillies had lower luteinizing hormone (LH) peak (8 ± 1 ng/ml) and basal (4 ± 0.5 ng/ml) levels, lower peak magnitude (2 ± 0.2 ng/ml) and period average (5 ± 0.6 ng/ml) than OV fillies (32 ± 4.5, 8 ± 1.2, 17.1 ± 6, and 15 ± 2.3 ng/ml, respectively). The PP group, in contrast to the OV group, showed no relationship between FSH surge and follicle wave emergence. We conclude that an LH concentration higher than 8 ng/ml is needed for follicle growth to a preovulatory size. Wave emergence and FSH secretion seem to be independent events, probably due to an inhibitory neural system in these PP animals. PP fillies may provide a physiological model for the study of follicle wave emergence which apparently does not depend on gonadotropin levels.

Effects of melatonin on the ovarian response to pinealectomy or continuous light in female rats: similarity with polycystic ovary syndrome

The current study was conducted to investigate the relationship between melatonin and chronic anovulation. Adult (3-4 months old) female Wistar rats were submitted to pinealectomy: group I: pinealectomized ovariectomized melatonin-treated (N = 10); group II: pinealectomized ovariectomized placebo-treated (N = 12); group III: pinealectomized light-treated placebo-treated(N = 10) or maintained under continuous light; group IV: maintained under continuous light, ovariectomized melatonin-treated (N = 22); group V: maintained under continuous light, ovariectomized placebo-treated (N = 10); group VI: maintained under continuous light placebo-treated (N = 10). In order to assess ovarian modifications, unilateral ovariectomy was performed during the fourth month in groups I, II, IV, V and the other ovary was removed after 8 months. Ovariectomy was performed in groups III and VI only after eight months. Melatonin (200 µg/100 g body weight) dissolved in 0.02 ml absolute ethanol was injected intramuscularly daily during the last 4 months into groups I and IV. The other groups were treated with placebo (NaCl). The ovarian cysts were analyzed and their area, perimeter and maximum diameter, as well as the thickness of the ovarian capsule were measured. Daily colpocytological smears were performed throughout the study. Persistent estrous condition and ovarian cysts were observed in all groups. In pinealectomized rats the ovarian and vaginal alterations disappeared at the end of the study and in rats maintained under continuous light the vaginal and ovarian polycystic aspect was reversed only in those treated with melatonin. We conclude that melatonin may act on the ovarian response reverting chronic anovulation induced by pinealectomy or continuous light.

Abnormal subcellular distribution of GLUT4 protein in obese and insulin-treated diabetic female dogs

The GLUT4 transporter plays a key role in insulin-induced glucose uptake, which is impaired in insulin resistance. The objective of the present study was to investigate the tissue content and the subcellular distribution of GLUT4 protein in 4- to 12-year-old control, obese and insulin-treated diabetic mongrel female dogs (4 animals per group). The parametrial white adipose tissue was sampled and processed to obtain both plasma membrane and microsome subcellular fractions for GLUT4 analysis by Western blotting. There was no significant difference in glycemia and insulinemia between control and obese animals. Diabetic dogs showed hyperglycemia (369.9 ± 89.9 mg/dl). Compared to control, the plasma membrane GLUT4, reported per g tissue, was reduced by 55% (P < 0.01) in obese dogs, and increased by 30% (P < 0.05) in diabetic dogs, and the microsomal GLUT4 was increased by ~45% (P < 0.001) in both obese and diabetic animals. Considering the sum of GLUT4 measured in plasma membrane and microsome as total cellular GLUT4, percent GLUT4 present in plasma membrane was reduced by ~65% (P < 0.001) in obese compared to control and diabetic animals. Since insulin stimulates GLUT4 translocation to the plasma membrane, percent GLUT4 in plasma membrane was divided by the insulinemia at the time of tissue removal and was found to be reduced by 75% (P < 0.01) in obese compared to control dogs. We conclude that the insulin-stimulated translocation of GLUT4 to the cell surface is reduced in obese female dogs. This probably contributes to insulin resistance, which plays an important role in glucose homeostasis in dogs.

Preliminary data on the prevalence of psychiatric disorders in Brazilian male and female juvenile delinquents

The aim of the present investigation was to study the prevalence of psychiatric disorders in a sample of delinquent adolescents of both genders and to compare the prevalence between genders. A total of 116 adolescents (99 males and 17 females) aged 12 to 19 on parole in the State of Rio de Janeiro were interviewed using the screening interview based on the Schedule for Affective Disorders and Schizophrenia for School-Age Children - Present and Lifetime (KSADS-PL). Data were collected between May 2002 and January 2003. Of 373 male and 58 female adolescents present in May 2002 in the largest institution that gives assistance to adolescents on parole in the city of Rio de Janeiro, 119 subjects were assessed (three of them refused to participate). Their average age was 16.5 years with no difference between genders. The screening interview was positive for psychopathology for most of the sample, with the frequencies of the suggested more prevalent psychiatric disorders being 54% for attention-deficit/hyperactivity disorder, 77% for conduct disorder, 41% for oppositional defiant disorder, 57% for anxiety disorder 57, 60% for depressive disorder 60, 63% for illicit drug abuse, and 58% for regular alcohol use. Internalizing disorders (depressive disorders, anxiety disorders and phobias) were more prevalent in the female subsample. There was no significant difference in the prevalence of illicit drug abuse between genders. There were more male than female adolescents on parole and failure to comply with the sentence was significantly more frequent in females. The high prevalence of psychopathology suggested by this study indicates the need for psychiatric treatment as part of the prevention of juvenile delinquency or as part of the sentence. However, treatment had never been available for 93% of the sample in this study.