Maternal hypothyroidism in mice influences glucose metabolism in adult offspring.

AIMS/HYPOTHESIS: During pregnancy, maternal metabolic disease and hormonal imbalance may alter fetal beta cell development and/or proliferation, thus leading to an increased risk for developing type 2 diabetes in adulthood. Although thyroid hormones play an important role in fetal endocrine pancreas development, the impact of maternal hypothyroidism on glucose homeostasis in adult offspring remains poorly understood. METHODS: We investigated this using a mouse model of hypothyroidism, induced by administration of an iodine-deficient diet supplemented with propylthiouracil during gestation. RESULTS: Here, we show that, when fed normal chow, adult mice born to hypothyroid mothers were more glucose-tolerant due to beta cell hyperproliferation (two- to threefold increase in Ki67-positive beta cells) and increased insulin sensitivity. However, following 8 weeks of high-fat feeding, these offspring gained 20% more body weight, became profoundly hyperinsulinaemic (with a 50% increase in fasting insulin concentration), insulin-resistant and glucose-intolerant compared with controls from euthyroid mothers. Furthermore, altered glucose metabolism was maintained in a second generation of animals. CONCLUSIONS/INTERPRETATION: Therefore, gestational hypothyroidism induces long-term alterations in endocrine pancreas function, which may have implications for type 2 diabetes prevention in affected individuals.

Th e eff ects of Nigella Sativa extract on renal tissue oxidative damage during neonatal and juvenile growth in propylthiouracil-induced hypothyroid rats.

OBJECTIVE: We investigated the effects of hydroalcoholic extract of Nigella sativa (NS) on renal tissue oxidative damage associated with propylthiouracil (PTU)-induced hypothyroidism during neonatal and juvenile growth in rats. METHODS: Pregnant rats were divided into five groups designated as: 1) control; 2) propylthiouracil (PTU); 3) PTU-NS100; 4) PTU-NS200, and 5) PTU-NS400. All mothers except the control group received 0.005% PTU in their drinking water during lactation. Besides PTU, mothers in groups 3-5 received 100, 200, and 400 mg/kg of NS extract. After lactation period, the off spring continued to receive the same experimental treatment for the first 8 weeks of their life. Ten male off springs of each group were randomly selected, blood samples collected, and the kidney tissues removed. RESULTS: The serum thyroxin concentration in PTU group was lower than control group and improved by extract. PTU increased the renal malondialdehyde (MDA), while reduced the total thiols concentrations and catalase (CAT) and superoxide dismutase (SOD) activity compared to control group. Administration of 200 and 400 mg/kg of NS extract decreased MDA level, while it increased the total thiols and 400 mg/kg increased CAT and SOD activity in renal tissues compared to PTU group. Serum creatinine and blood urea nitrogen (BUN) in PTU group was higher than in comparison with the control group. 400 mg/kg decreased creatinine, but both 200 and 400 mg/kg improved BUN concentration compared to PTU group. CONCLUSION: The results of this study demonstrate that the hydroalcoholic extract of NS has a protective effect on the renal tissue oxidative damage associated with PTU-induced hypothyroidism during neonatal and juvenile growth in rats.

Opposing Effects of Maternal Hypo- and Hyperthyroidism on the Stability of Thalamocortical Synapses in the Visual Cortex of Adult Offspring.

Insufficient or excessive thyroid hormone (TH) levels during fetal development can cause long-term neurological and cognitive problems. Studies in animal models of perinatal hypo- and hyperthyroidism suggest that these problems may be a consequence of the formation of maladaptive circuitry in the cerebral cortex, which can persist into adulthood. Here we used mouse models of maternal hypo- and hyperthyroidism to investigate the long-term effects of altering thyroxine (T4) levels during pregnancy (corresponding to embryonic days 6.5-18.5) on thalamocortical (TC) axon dynamics in adult offspring. Because perinatal hypothyroidism has been linked to visual processing deficits in humans, we performed chronic two-photon imaging of TC axons and boutons in primary visual cortex (V1). We found that a decrease or increase in maternal serum T4 levels was associated with atypical steady-state dynamics of TC axons and boutons in V1 of adult offspring. Hypothyroid offspring exhibited axonal branch and bouton dynamics indicative of an abnormal increase in TC connectivity, whereas changes in hyperthyroid offspring were indicative of an abnormal decrease in TC connectivity. Collectively, our data suggest that alterations to prenatal T4 levels can cause long-term synaptic instability in TC circuits, which could impair early stages of visual processing.

Synergic actions of polyphenols and cyanogens of peanut seed coat (Arachis hypogaea) on cytological, biochemical and functional changes in thyroid.

In animals, long-term feeding with peanut (Arachis hypogaea) seed coats causes hypertrophy and hyperplasia of the thyroid gland. However, to date there have been no detailed studies. Here, we explored the thyroidal effects of dietary peanut seed coats (PSC) in rats. The PSC has high levels of pro-goitrogenic substances including phenolic and other cyanogenic constituents. The PSC was mixed with a standard diet and fed to rats for 30 and 60 days, respectively. Animals fed with the PSC-supplemented diet showed a significant increase in urinary excretion of thiocyanate and iodine, thyroid enlargement, and hypertrophy and/or hyperplasia of thyroid follicles. In addition, there was inhibition of thyroid peroxidase (TPO) activity, 5'-deiodinase-I (DIO1) activity, and (Na+-K+)-ATPase activity in the experimental groups of rats as compared to controls. Furthermore, the PSC fed animals exhibited decreased serum circulating total T4 and T3 levels, severe in the group treated for longer duration. These data indicate that PSC could be a novel disruptor of thyroid function, due to synergistic actions of phenolic as well as cyanogenic constituents.

Irreversible damage to auditory system functions caused by perinatal hypothyroidism in rats.

We examined the effect of perinatal hypothyroidism on auditory function in rats using a prepulse inhibition paradigm. Pregnant rats were treated with the antithyroid drug methimazole (1-methyl-2-mercaptoimidazole) from gestational day 15 to postnatal day 21 via drinking water at concentrations (w/v) of 0 (control), 0.002 (low dose), or 0.02% (high dose). Rats from methimazole-treated mothers were tested at ages 1, 6, and 12months using techniques to examine prepulse inhibition and startle response. The startle stimulus consisted of 40ms of white noise at 115dB, whereas the prepulse, which preceded the startle stimulus by 30ms, consisted of 20ms of white noise at 75, 85, or 95dB. When the prepulse intensity was 75 or 85dB, the high-dose group showed decreased prepulse inhibition percentages compared with the control and low-dose groups. The reduced percentages of prepulse inhibition did not return to control levels over the 12-month study period. In contrast, no differences in prepulse inhibition were observed among the three dose groups when prepulse intensity was 95dB. Moreover, the high-dose group displayed excessive reaction to auditory startle stimuli compared with the other groups. Reductions in plasma free thyroxine and body weight gain were observed in the high-dose group. We conclude that perinatal hypothyroidism results in irreversible damage to auditory function in rats.

Toxicity of methimazole on femoral bone in suckling rats: alleviation by selenium.

AIMS: Selenium has a pharmacological properties and it is well considered as an antioxidant. The present study investigated the potential ability of selenium, used as a nutritional supplement, to alleviate bone impairments in suckling rats whose mothers were treated with methimazole, an antithyroid drug. MAIN METHODS: Female Wistar rats were randomly divided into four groups of six each: group I served as control which received standard diet; group II were rendered hypothyroid by administration of methimazole (250 mg L(-1) in their drinking water); group III received both methimazole (250 mg L(-1) in their drinking water) and selenium (0.5 mg kg(-1) of diet); group IV received 0.5 Na(2)SeO(3) mg kg(-1) of diet. Treatments were started from the 14th day of pregnancy until day 14 after delivery. KEY FINDINGS: Methimazole treatment decreased femur length and weight in 14-day-old rats, when compared to controls. Femur antioxidant enzyme activities, superoxide dismutase, catalase and glutathione peroxidase decreased. Lipid peroxidation recorded an increase revealed by high femur malondialdehyde levels. Methimazole also caused a significant decrease in calcium and phosphorus levels in bone. Yet, in plasma and urine, they increased and decreased inversely. Besides, plasma total tartrate-resistant acid phosphatase was enhanced, while total alkaline phosphatase was reduced. Co-administration of selenium through diet improved the biochemical parameters cited above. Nevertheless, distorted histoarchitecture revealed in hypothyroid rat femur was alleviated by Se treatment. SIGNIFICANCE: The present study suggests that selenium is an important protective element that may be used as a dietary supplement protecting against bone impairments.

Induction of oxidative stress and inhibition of superoxide dismutase expression in rat cerebral cortex and cerebellum by PTU-induced hypothyroidism and its reversal by curcumin.

The present study was carried out to elucidate the effectiveness of curcumin in ameliorating the expression of superoxide dismutase (SOD) in cerebral cortex and cerebellum of rat brain under 6-propyl-2-thiouracil (PTU)-induced hypothyroidism. Induction of hypothyroidism in adult rats by PTU resulted in augmentation of lipid peroxidation (LPx), an index of oxidative stress in cerebellum but not in cerebral cortex. Curcumin-supplementation to PTU-treated (hypothyroid) rats showed significant reduction in the level of LPx in both the regions of brain. The decreased translated products (SOD1 and SOD2) and the unchanged activity of SOD in cerebral cortex of PTU-treated rats were increased on supplementation of curcumin to the hypothyroid rats. Declined translated products of SOD1 and SOD2 in cerebellum of PTU-treated rats were alleviated on administration of curcumin to hypothyroid rats. On the other hand, the decreased activity of SOD in cerebellum of PTU-treated rats was further declined on administration of curcumin to the hypothyroid rats. Results of the present investigation indicate that curcumin differentially modulates the expression of superoxide dismutase in rat brain cortex and cerebellum under PTU-induced hypothyroidism.

Interlaboratory study comparison of the 15-day intact adult male rat screening assay: evaluation of an antithyroid chemical and a negative control chemical.

Validation of the 15-day intact adult male rat screening assay (IAMRSA), an endocrine activity screen, was extended beyond the 28 substances evaluated to date. Two independent laboratories evaluated specificity using allyl alcohol (AA), a putative negative control, and DE-71 (technical grade pentabromodiphenyl ether) for comparison with previous pubertal assays that demonstrated thyroid effects. Male rats (15/group) were gavaged daily with AA (0, 10, 30, or 40 mg/kg/day) or DE-71 (0, 3, 30, or 60 mg/kg/day) for 15 days. Body and organ weights and serum hormone concentrations were measured, and a limited histopathological assessment was conducted. AA results were considered negative at doses that did not exceed the maximum tolerated dose (MTD); effects reported were dose-related decreases in weight gain, increased liver weights and, although the pattern varied across studies, alterations in some androgen-sensitive endpoints in the high-dose where the maximum tolerated dose was exceeded. In the DE-71 studies, dose-dependent increases in liver weights (consistent with hepatic enzyme induction), decreases in tri-iodothyronine and thyroxine, concomitant thyroid stimulating hormone increases were observed and one laboratory reported histopathological thyroid changes in mid- and high-dose groups, and the other increased thyroid weights. For DE-71, the IAMRSA was comparable in sensitivity to the pubertal assays. Overall, the specificity and sensitivity of the IAMRSA for deployment in an endocrine screening battery are supported. However, differentiating primary endocrine-mediated effects from secondary effects caused by systemic toxicity will be challenging, emphasizing the need to utilize a battery of assays and a weight of evidence approach when evaluating the potential endocrine activity of chemicals.

Protective effects of selenium on methimazole-induced anemia and oxidative stress in adult rats and their offspring.

The present study investigates the potential ability of selenium, considered as an antioxidant with pharmacological property to alleviate oxidative stress and hematological parameter disorders induced by methimazole, an antithyroid drug. Pregnant Wistar rats were randomly divided into four groups of six each: group I served as negative control and received a standard diet; group II received 250 mg/L of methimazole in drinking water and a standard diet; group III received both methimazole (250 mg/L, orally) and selenium (0.5 mg/kg of diet) supplemented to the standard diet; group IV served as positive control and received a supplement of selenium in the diet (0.5 mg/kg of diet) as sodium selenite (Na(2)SeO(3)). Treatment was started from the 14th day of pregnancy until day 14 after delivery. Methimazole reduced the number of red blood cells, hemoglobin concentration and hematocrit in mothers and their pups. Besides, plasma iron, vitamins B(9), B(12), C and E levels were reduced. Lipid peroxidation increased, objectified by high malondialdehyde levels and lactate dehydrogenase activity in plasma, while glutathione, glutathione peroxidase, superoxide dismutase and catalase activities showed a significant decline. Co-administration of selenium through diet improved all the parameters cited above. It can be concluded that the administration of selenium alleviates methimazole-induced toxicity, thus demonstrating its antioxidant efficacy.

Protective effects of selenium on methimazole nephrotoxicity in adult rats and their offspring.

This study aims to investigate the improving effects of selenium on methimazole-induced kidney impairments in adult rats and their pups. The animals were randomly divided into four groups of six each: group I served as control which received standard diet; group II received only methimazole in drinking water as 250 mg/l; group III received both methimazole (250 mg/l, orally) and selenium (0.5 mg/kg of diet); group IV served as a positive control and received selenium (0.5 mg/kg of diet) as sodium selenite (Na(2)SeO(3)). Treatments were started from the 14th day of pregnancy until day 14 after delivery. In the methimazole-treated group, body and absolute kidney weights decreased in pups and their mothers when compared to control. Daily urine volume, plasma creatinine levels were higher, while urinary levels were lower than in control. Besides, antioxidant enzyme activities, superoxide dismutase, catalase and glutathione peroxidase decreased. Lipid peroxidation recorded an increase revealed by high kidney malondialdehyde levels, while those of plasma and urinary uric acid showed a significant decline. Methimazole-treated rat kidneys exhibited leucocytic infiltrations, vascular congestion and narrowed Bowman's space. Co-administration of selenium through diet improved all the parameters cited above in adult rats and their progeny. Nevertheless, the distorted histoarchitecture in rat kidney was alleviated by selenium treatment. It can then be concluded that selenium is an important protective element that may be used as a dietary supplement against kidney impairments.

Goitrogenic/antithyroidal potential of green tea extract in relation to catechin in rats.

Catechins are flavonoids found in abundance in green tea, have elicited high interest due to their beneficial effects on health. Though flavonoids have been reported to have an antithyroid effect and also to be goitrogenic there have been no reports about the effect of green tea on rat thyroid. The present study was designed to examine whether high doses of green tea has any harmful effect on thyroid physiology. For this purpose green tea extract was administered orally to male albino rats for 30 days at doses of 1.25 g%, 2.5 g% and 5.0 g%, respectively. Similarly, pure catechin was administered at doses of 25, 50 and 100mg/kg body weight which is equivalent to above doses of green tea extract. Lower body weight gain associated with marked hypertrophy and/or hyperplasia of the follicles was noted in the high dose of green tea and catechin treated groups. Decreased activity of thyroid peroxidase and 5'-deiodinase I and substantially elevated thyroidal Na,K+ATPase activity have been observed. Moreover, serum T3 and T4 levels were found to reduce followed by significant elevation of serum TSH. Taken together, these results suggest that catechin present in green tea extract might behave as antithyroid agent and possibly the consumption of green tea at high dose could alter thyroid function adversely.

Dietary selenium addition improves cerebrum and cerebellum impairments induced by methimazole in suckling rats.

The aim of this study is to investigate the improving effects of selenium (Se) on cerebrum and cerebellum impairments induced by methimazole (MMI) in suckling rats. Animals were randomly divided into four groups of six each: group I served as control which received standard diet; group II received only MMI (250 mg L(-1)(,) orally); group III received both MMI (250 mg L(-1), orally) and Se (0.5 mg kg(-1) of diet); group IV served as a positive control and received Se (0.5 mg kg(-1) of diet) as sodium selenite (Na(2)SeO(3)). Treatments were started from the 14th day of pregnancy until day 14 after delivery. In the MMI-treated group, plasma-free thyroid hormone levels (FT(3) and FT(4)), protein, DNA and RNA contents in cerebrum and cerebellum decreased when compared to control. Co-treatment with Se ameliorated these parameters. In the MMI-treated group, antioxidant enzyme activities (superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px) significantly decreased, while malonaldialdehyde (MDA) levels in cerebrum and cerebellum increased. Co-administration of Se through the diet restored these parameters to near normal values. The biochemical modifications are correlated histologically with the abnormal development of an external granular layer, indicating a delay of granular cells migration towards the molecular layer in the MMI-treated group. Our results showed that Se improved cerebrum and cerebellum MMI-induced damages in suckling rats. Moreover, we concluded that Se is an important neuroprotective element that may be used as a dietary supplement against brain impairments.

Protective role of Mangifera indica, Cucumis melo and Citrullus vulgaris peel extracts in chemically induced hypothyroidism.

An investigation was made to evaluate the pharmacological importance of fruit peel extracts of Mangifera indica (MI), Citrullus vulgaris (CV) and Cucumis melo (CM) with respect to the possible regulation of tissue lipid peroxidation (LPO), thyroid dysfunctions, lipid and glucose metabolism. Pre-standardized doses (200mg/kg of MI and 100mg/kg both of CV and CM), based on the maximum inhibition in hepatic LPO, were administered to Wistar albino male rats for 10 consecutive days and the changes in tissue (heart, liver and kidney) LPO and in the concentrations of serum triiodothyronine (T(3)), thyroxin (T(4)), insulin, glucose, alpha-amylase and different lipids were examined. Administration of three test peel extracts significantly increased both the thyroid hormones (T(3) and T(4)) with a concomitant decrease in tissue LPO, suggesting their thyroid stimulatory and antiperoxidative role. This thyroid stimulatory nature was also exhibited in propylthiouracil (PTU) induced hypothyroid animals. However, only minor influence was observed in serum lipid profile in which CM reduced the concentrations of total cholesterol and low-density lipoprotein-cholesterol (LDL-C), while CV decreased triglycerides and very low-density lipoprotein-cholesterol (VLDL-C). When the combined effects of either two (MI+CV) or three (MI+CV+CM) peel extracts were evaluated in euthyroid animals, serum T(3) concentration was increased in response to MI+CV and MI+CV+CM treatments, while T(4) level was elevated by the combinations of first two peels only. Interestingly, both the categories of combinations increased T(4) levels, but not T(3) in PTU treated hypothyroid animals. Moreover, a parallel increase in hepatic and renal LPO was observed in these animals, suggesting their unsafe nature in combination. In conclusion the three test peel extracts appear to be stimulatory to thyroid functions and inhibitory to tissue LPO but only when treated individually.

Autism: transient in utero hypothyroxinemia related to maternal flavonoid ingestion during pregnancy and to other environmental antithyroid agents.

The incidence and prevalence of autism have increased during the past two decades. Despite comprehensive genetic studies the cause of autism remains unknown. This review emphasizes the potential importance of environmental factors in its causation. Alterations of cortical neuronal migration and cerebellar Purkinje cells have been observed in autism. Neuronal migration, via reelin regulation, requires triiodothyronine (T3) produced by deiodination of thyroxine (T4) by fetal brain deiodinases. Experimental animal models have shown that transient intrauterine deficits of thyroid hormones (as brief as 3 days) result in permanent alterations of cerebral cortical architecture reminiscent of those observed in brains of patients with autism. I postulate that early maternal hypothyroxinemia resulting in low T3 in the fetal brain during the period of neuronal cell migration (weeks 8-12 of pregnancy) may produce morphological brain changes leading to autism. Insufficient dietary iodine intake and a number of environmental antithyroid and goitrogenic agents can affect maternal thyroid function during pregnancy. The most common causes could include inhibition of deiodinases D2 or D3 from maternal ingestion of dietary flavonoids or from antithyroid environmental contaminants. Some plant isoflavonoids have profound effects on thyroid hormones and on the hypothalamus-pituitary axis. Genistein and daidzein from soy (Glycine max) inhibit thyroperoxidase that catalyzes iodination and thyroid hormone biosynthesis. Other plants with hypothyroid effects include pearl millet (Pennisetum glaucum) and fonio millet (Digitaria exilis); thiocyanate is found in Brassicae plants including cabbage, cauliflower, kale, rutabaga, and kohlrabi, as well as in tropical plants such as cassava, lima beans, linseed, bamboo shoots, and sweet potatoes. Tobacco smoke is also a source of thiocyanate. Environmental contaminants interfere with thyroid function including 60% of all herbicides, in particular 2,4-dichlorophenoxyacetic acid (2,4-D), acetochlor, aminotriazole, amitrole, bromoxynil, pendamethalin, mancozeb, and thioureas. Other antithyroid agents include polychlorinated biphenyls (PCBs), perchlorates, mercury, and coal derivatives such as resorcinol, phthalates, and anthracenes. A leading ecological study in Texas has correlated higher rates of autism in school districts affected by large environmental releases of mercury from industrial sources. Mercury is a well known antithyroid substance causing inhibition of deiodinases and thyroid peroxidase. The current surge of autism could be related to transient maternal hypothyroxinemia resulting from dietary and/or environmental exposure to antithyroid agents. Additional multidisciplinary epidemiological studies will be required to confirm this environmental hypothesis of autism.

Ameliorative effect of vitamins (alpha-tocopherol and ascorbic acid) on PCB (Aroclor 1254) induced oxidative stress in rat epididymal sperm.

The aim of this study was to investigate the possible protective role of vitamins on PCB (Aroclor 1254)-induced spermiotoxicity using qualitative, quantitative and biochemical approaches. Adult male albino rats of Wistar strain were randomly divided into four groups, each group consists of six animals. The control group received corn oil, the second group of rats were administered Aroclor 1254 at a dose of 2 mg/kg bw/day intraperitoneally for 30 days. The third group of rats were treated with Aroclor 1254 along with alpha-tocopherol (50 mg/kg of bw/day) for 30 days, while the fourth group of rats were treated with Aroclor 1254 along with ascorbic acid (100 mg/kg bw/day) orally for 30 days. Twenty-four hours after the last treatment, control and experimental animals were killed by decapitation. Sperm was collected from the cauda epididymal region and its count and motility were detected. Sperm was sonicated and used for the estimation of reactive oxygen species (ROS) [hydroxyl radical (HO(*)) and hydrogen peroxide (H(2)O(2))], non-enzymic antioxidants [alpha-tocopherol, ascorbic acid and reduced glutathione (GSH)], activity of enzymic antioxidants [superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) glutathione reductase (GR) and glutathione-S-transferase (GST)] and lipid peroxidation (LPO). The result of this experiment shows that PCB significantly decreases the level of alpha-tocopherol, ascorbic acid and GSH and the activities of SOD, CAT, GPx, GR and GST with elevated levels of ROS and LPO. In addition, decreased epididymal sperm motility and count were observed. Simultaneous supplementation with alpha-tocopherol and ascorbic acid restored these parameters to that of normal range. In conclusion, alpha-tocopherol and ascorbic acid exhibited protective effect on sperm by inhibiting PCB-induced ROS generation.

Role of alpha-tocopherol on antioxidant status in liver, lung and kidney of PCB exposed male albino rats.

Polychlorinated biphenyls (PCB) are widespread, lipophilic environmental pollutants which have been identified as contaminants in almost every component of the global ecosystem including fish, wildlife, and human adipose tissue, breast milk, and serum. Several studies have shown that PCBs can cause oxidative damage to biomolecules, in the form of lipid peroxidation, modulation of antioxidant enzymes, and oxidative stress. In the present study, we have sought to investigate the effects of alpha-tocopherol (vitamin E) on antioxidant status of PCB-induced toxicity in male Wistar rats. The protective effect of alpha-tocopherol (50mg/kg body weight/day) was tested in PCB-induced toxicity in rat liver, lung, and kidney. We report here that the oral supplementation of alpha-tocopherol was found to maintain the cellular redox status by maintaining the activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase accompanied with glutathione and vitamin E levels and down regulation in the levels of lipid peroxides, hydroxyl radical and hydrogen peroxides generation in PCB treated rats. Therefore, our present study demonstrates the PCB-induced deficits in antioxidant enzyme activities and increase in reactive oxygen species and lipid peroxidation levels in liver, kidney, and lung which can be overcome through simultaneous supplementation with alpha-tocopherol.

Prenatal Aroclor 1254 exposure and brain sexual differentiation: effect on the expression of testosterone metabolizing enzymes and androgen receptors in the hypothalamus of male and female rats.

Polychlorinated biphenyls (PCBs) are industrial pollutants detected in human milk, serum and tissues. They readily cross the placenta to accumulate in fetal tissues, particularly the brain. These compounds affect normal brain sexual differentiation by mechanisms that are incompletely understood. The aim of this study was to verify whether a technical mixture of PCBs (Aroclor 1254) would interfere with the normal pattern of expression of hypothalamic aromatase and 5-alpha reductase(s), the two main enzymatic pathways involved in testosterone activation and of androgen receptor (AR). Aroclor 1254 was administered to pregnant rats at a daily dose of 25 mg/kg by gavage from days 15 to 19 of gestation (GD15-19). At GD20 the expression of aromatase, 5-alpha reductase types 1 and 2 and androgen receptor (AR) and aromatase activity were evaluated in the hypothalamus of male and female embryos. The direct effect of Aroclor was also evaluated on aromatase activity adding the PCB mixture to hypothalamic homogenates or to primary hypothalamic neuronal cultures. The data indicate that aromatase expression and activity is not altered by prenatal PCB treatment; 5-alpha reductase type 1 is similarly unaffected while 5-alpha reductase type 2 is markedly stimulated by the PCB exposure in females. Aroclor also decreases the expression of the AR in females. The observed in vivo effects are indicative of a possible adverse effect of PCBs on the important metabolic pathways by which testosterone produces its brain effects. In particular the changes of 5-alpha reductase type 2 and AR in females might be one of the mechanisms by which Aroclor exposure during fetal development affects adult sexual behavior in female rats.

Protective effect of quercetin on aroclor 1254-induced oxidative damage in cultured chicken spermatogonial cells.

Quercetin, a dietary-derived falvonol-type flavonoid, is ubiquitous in fruits and vegetables and plays important roles in human health by virtue of its antioxidant function. The present study was performed to investigate effects of quercetin on oxidative damage that was induced by an environmental endocrine disrupter, Aroclor 1254 (A1254), in cultured spermatogonial cells of embryonic chickens. Spermatogonial cells were dispersed from 18-day-old embryo and exposed to A1254 alone or in combination with quercetin. The oxidative damage was estimated by measuring contents of thiobarbituric acid-reactive substances (TBARS, an indicator of lipid peroxidation), activity of superoxide dismutase (SOD, a scavenger of superoxide), and activity of glutathione (GSH, an intracellular antioxidant). Results showed that quercetin had no deleterious effect on spermatogonial cells at 0.01 approximately 1 microg/ml. Exposure to A1254 (10 microg/ml) induced an increase of spermatogonial cell number, and membrane integrity was damaged by elevation of lactate dehydrogenase (LDH) leakage. Exposure to A1254 also induced an elevation in TBARS but a decrease in SOD activity and GSH content. However, compared with A1254 treatment alone, simultaneous supplementation with quercetin decreased LDH leakage to maintain the cell integrity, decreased the levels of TBARS to quench the free radicals, increased SOD activity and GSH content to restore the endogenous antioxidant defense system. Thus, quercetin displayed protective effects on spermatogonial cells from A1254-induced oxidative damage through increasing intracellular antioxidant levels and decreasing lipid peroxidation. Consequently, the antioxidant, such as quercetin, from food or feed consumed by human and animals may attenuate the negative effects of environmental endocrine disrupters.

Repeated exposure of adult rats to Aroclor 1254 causes brain region-specific changes in intracellular Ca2+ buffering and protein kinase C activity in the absence of changes in tyrosine hydroxylase.

Polychlorinated biphenyls (PCBs) are ubiquitous environmental contaminants, some of which may be neurotoxic. In vitro studies from this laboratory indicated that noncoplanar PCBs perturbed intracellular signal transduction mechanisms including Ca2+ homeostasis, receptor-mediated inositol phosphate production, and translocation of protein kinase C (PKC). In the present study, we examined the effects of PCBs in vivo by dosing adult male Long-Evans rats orally with Aroclor 1254 (0, 10, or 30 mg/kg/day; 5 days/week for 4 weeks) in corn oil. At 24 h after the last dose, rats were tested for motor activity in a photocell device for 30 min. Immediately, the rats were euthanized, blood was collected for thyroid hormone analysis, and brains were removed, dissected into regions (cerebellum, frontal cortex, and striatum), and subcellular fractions were obtained for neurochemical analysis. Following Aroclor 1254 treatment, body weight gain in the high-dose group was significantly lower than the control and low-dose groups. Horizontal motor activity was significantly lower in rats dosed with 30 mg/kg Aroclor 1254. Ca2+ buffering by microsomes was significantly lower in all three brain regions from the 30 mg/kg group. In the same dose group, mitochondrial Ca2+ buffering was affected in cerebellum but not in cortex or striatum. Similarly, total cerebellar PKC activity was decreased significantly while membrane-bound PKC activity was significantly elevated at 10 and 30 mg/kg. PKC activity was not altered either in cortex or the striatum. Neurotransmitter levels in striatum or cortex were slightly altered in PCB-exposed rats compared to controls. Furthermore, repeated oral administration of Aroclor 1254 to rats did not significantly alter forebrain tyrosine hydroxylase immunoreactivity or enzymatic activity. Circulating T4 (total and free) concentrations were severely depressed at both doses in Aroclor 1254-exposed rats compared to control rats, suggesting a severe hypothyroid state. These results indicate that (1) in vivo exposure to a PCB mixture can produce changes in second messenger systems that are similar to those observed after in vitro exposure of neuronal cell cultures; (2) second messenger systems seem to be more sensitive than alterations in neurotransmitter levels or tyrosine hydroxylase involved in dopamine synthesis during repeated exposure to PCBs; and (3) the observed motor activity changes were independent of changes in striatal dopamine levels.

Validation of automated behavioral test systems.

A positive control study was conducted as part of the ongoing validation program for developmental neurotoxicity testing in our laboratory using a standard battery of automated systems, consisting of rotorod, motor activity, acoustic startle, and two-way active avoidance. Female Sprague-Dawley rats were given 10 mg/kg diazepam (DZ) by SC injection or 20 mg/kg methimazole (MET) by gavage from gestation day 15 (DZ) or 17 (MET) through postpartum day 10; a group of control animals remained untreated. Offspring were assessed for growth, survival, developmental landmarks, and behavior. Although this study was considered useful for obtaining historical data, it offered few advantages in terms of validation of automated behavior test systems. Perinatal treatment with DZ resulted in no maternal toxicity and no adverse effects on growth or development of F1 offspring; a deficit in acoustic startle responding was the only behavioral effect observed. Treatment with MET resulted in maternal toxicity, reduced neonatal body weights, and developmental delays. Behavioral effects included impaired rotorod performance and acoustic startle responding (neonates), and enhanced motor activity and acoustic startle responding (young adults). However, effects on shuttle avoidance were not observed for either drug, and only one direction of behavioral effect occurred for the rotorod and motor activity systems. These results, as well as those from subsequent studies in our laboratory, suggest that it may be preferable to validate automated behavior systems using short-term studies in which young adult animals are treated directly with positive control agents.