Investigation of protein-styrene oxide adducts as a molecular biomarker of human exposed to styrene.

Hemoglobin-styrene oxide adducts in blood have been studied as a molecular biomarker of worker exposed to styrene. Determination of protein-styrene oxide adducts in different biological samples with modified Raney-Ni procedure is described in this paper. The following biological samples have been investigated: fresh rat blood reacted with styrene oxide in vitro; rat blood reacted with styrene or styrene oxide in vivo; vein blood from workers exposed to styrene in two factories. The data showed that there was a good linear dose-response relationship between reacting dose of styrene oxide or styrene and amount of protein-styrene oxide adducts in both in vitro and in vivo experiments. For human samples, a dose-response relationship between protein adducts and styrene exposure can be found in glass fiber factory, but not in piano manufacture plant.

Insulin-like growth factor I inhibits aromatization induced by follice-stimulating hormone in rat sertoli cell culture.

Sertoli cells in the testis and granulosa cells in the ovary convert androgen to estrogen under the primary control of FSH. Insulin-like growth factor I (IGF-I) markedly augments FSH-stimulated estrogen production in the rat granulosa cell. In this study we examined the regulation of aromatase by FSH and characterized the effects of IGF-I on FSH-induced estrogen production by Sertoli cells cultured from the testes of 16-day-old rats. FSH stimulated aromatization of androstenedione in Sertoli cell culture and achieved maximal effectiveness by 12 h of treatment. Analysis of aromatase mRNA by reverse transcription-polymerase chain reaction indicated a marked induction by FSH within 3 h of treatment that was dependent on FSH concentration. IGF-I inhibited FSH-stimulated aromatization dose-dependently; inhibition was approximately 50% by 6 h of cotreatment (p < 0.01). IGF-I was ineffective if added more than 3 h after addition of FSH. Aromatase mRNA was reduced by IGF-I (37 +/- 12%, p < 0.01), coincident with the decrease in estrogen production. To further address the mechanism of IGF-I inhibition, potential interactions with the cAMP and protein kinase C (PKC) signaling pathways were examined. IGF-I inhibited aromatase activity induced by dibutyryl cAMP and inhibited FSH-stimulated estrogen production in the presence of 3-isobutyl-1-methylxanthine, suggesting that IGF-I action was independent of cAMP production. Phorbol-12-myristate-13-acetate (PMA) and IGF-I were additive in their inhibition of FSH. However, down-regulation of PKC prevented PMA inhibition of FSH but not inhibition by IGF-I. We conclude that IGF-I specifically inhibits FSH-induced aromatization in the Sertoli cell in marked contrast to the effects of IGF-I on rat granulosa cells. Although IGF-I and PMA both inhibit aromatase induction, the independence of the IGF-I effect from PKC down-regulation suggests that the initial action of IGF-I is independent of PKC. As IGF-I treatment similarly alters FSH stimulation of both estrogen production and aromatase mRNA, it is likely that the effect of IGF-I on estrogen production in the Sertoli cell is a result, at least in part, of a decrease in aromatase mRNA.

Insulin-like growth factor (IGF) binding protein 3 in the rat testis: follicle-stimulating hormone dependence of mRNA expression and inhibition of IGF-I action on cultured Sertoli cells.

Insulin-like growth factor binding protein 3 (IGFBP-3) is the predominant IGF binding protein produced by cultured rat Sertoli cells. Previous studies have shown that FSH lowers the abundance of IGFBP-3 protein in Sertoli cell culture medium, suggesting that this binding protein has a physiological role in modulating insulin-like growth factor-I (IGF-I) activity in the testis. To characterize the physiological relevance of the FSH regulation of IGFBP-3, in this study we examined effects of FSH on IGFBP-3 mRNA expression in testes from hypophysectomized rats and in Sertoli cell culture. We then examined whether or not IGFBP-3 could alter the effects of IGF-I on cultured Sertoli cells. FSH (1-300 ng/ml) treatment of rat Sertoli cells dose-dependently decreased IGFBP-3 mRNA, with near-complete inhibition by 12 h of treatment. To evaluate this effect in the whole animal, male rats were hypophysectomized at 20 days of age and injected with FSH 10 days later. Testis IGFBP-3 mRNA increased following hypophysectomy relative to the value in sham animals, while FSH treatment decreased IGFBP-3 mRNA to sham levels within 6 h. To determine whether or not IGFBP-3 modulates IGF-I activity in the Sertoli cell, recombinant human non-glycosylated rIGFBP-3 was added to Sertoli cell cultures in the presence or absence of IGF-I. The rIGFBP-3 dose-dependently inhibited IGF-I-stimulated lactate production with a half-maximal dose of 100 ng/ml. Neither basal lactate production nor FSH-stimulated lactate was altered by addition of rIGFBP-3.(ABSTRACT TRUNCATED AT 250 WORDS)

Heterogeneous distribution of D1, D2 and D5 receptor mRNAs in monkey striatum.

The primate striatum has a compartmental organization reflected both in the topography of its afferent projections and in the segregation of its morphologically similar but neurochemically distinct efferent neurons. Discretely projecting mesostriatal neurons release dopamine (DA) which modulates the responses of striatal neurons to other afferent inputs. Multiple DA receptor (DAR) subtypes have been cloned and characterized and mapping their cellular expression is crucial for understanding the influence of DA on striatal function. We report the distribution of mRNAs for D1, D2 and D5 DAR subtypes (D2R, D2R and D5R) in the striatum of cynomolgus monkeys (Macaca fascicularis) studied by in situ hybridization histochemistry (ISH) using monkey-specific cRNA probes. Adjacent sections were stained for calbindin immunoreactivity to distinguish striosomal and matrix compartments for comparison with the patterns obtained with ISH. In the caudate nucleus, D1R mRNA was concentrated in calbindin-poor striosomes where dense grain clusters were seen overlying the majority of medium-sized neurons (diameter approximately 15 microns). D1R mRNA localization was relatively homogeneous in the putamen. By contrast, the distributions of D2R and D5R mRNAs showed no clear preference for the striosomal or matrix compartments of either caudate nucleus or putamen. In the ventral striatum (nucleus accumbens, olfactory tubercle and ventral portions of caudate nucleus and putamen), expression of D1R and D2R mRNA was sparse relative to dorsal striatum, while D5R mRNA expression was roughly equal in ventral and dorsal striatum. Circumscribed zones of hybridization associated with islands of tightly packed small cells occurred with all three DAR mRNA subtypes in the ventral striatum.(ABSTRACT TRUNCATED AT 250 WORDS)

Parathyroid hormone and calcitonin modify inositol phospholipid metabolism in fetal rat limb bones.

Inositol-containing phospholipids are believed to be intimately involved in the first steps of cellular signalling by certain hormones and neurotransmitters. We examined whether parathyroid hormone (PTH) and calcitonin (CT), two hormones that affect bone physiology, would elicit changes in inositol-phospholipid metabolism in cultured bone. [3H]inositol readily entered into the tissue phospholipid pool in fetal rat limb bones, and incorporated into phosphatidylinositol (92.9%), phosphatidylinositol-4-P (4.5%), and phosphatidylinositol-4,5-P2 (2.6%). PTH enhanced the incorporation of inositol into PtdIns in limb bones following 2- or 24-h hormone treatments. The effect of PTH was dose dependent (EC50 of 0.3-0.4 nM) and occurred in a concentration range similar to that for hormone-stimulated bone resorption. In contrast, 24-h treatment with CT-inhibited inositol incorporation, also in a dose-dependent manner. Two-hour CT treatment had variable effects on labeling. CT inhibited the stimulatory effect of PTH at both 2 and 24 h. The effects induced by PTH and CT were specific for PtdIns and were independent of the [3H]inositol pool size. These results indicate that inositol-phospholipid turnover can be modified during the action of these hormones on bone tissue. Although the time course of hormone-stimulated inositol incorporation observed here is slower than that found in other tissues, the change in phosphatidylinositol metabolism could mediate delayed effects of PTH or CT. Alternatively, alterations induced by PTH and CT in bone cell membranes, cell populations, or in the mineralized matrix could conceivably result in secondary changes in phosphatidylinositol metabolism.

Ametantrone inhibits prostaglandin--mediated resorption in bone organ culture.

Prostaglandins (PG) have been postulated to be involved in both tumor metastases to bone and in tumor-induced bone resorption. The anthracenedione antineoplastic agents ametantrone (HAQ) and mitoxantrone are potent antioxidants and inhibit hydroperoxide-dependent initiation and propagation reactions. Therefore, these compounds may inhibit PG production and could also inhibit tumor metastases and tumor-induced resorption. The ability of HAQ, a prototypic anthracenedione, to inhibit PG synthesis and PG-mediated bone resorption was investigated using neonatal mouse calvaria in organ culture. Epidermal growth factor (EGF) stimulates bone resorption in this tissue by inducing PG synthesis. Consequently, if HAQ inhibits EGF-stimulated PG synthesis, it should also inhibit EGF-stimulated bone resorption. HAQ, at 10 microM, completely abolished EGF-stimulated PG synthesis and calcium release. Moreover, HAQ (1.0-30 microM) inhibition of EGF-stimulated PGE2 synthesis correlated with the inhibition of EGF-stimulated Ca release in a concentration-dependent manner. In contrast to EGF, parathyroid hormone stimulates resorption by a PG-independent pathway. HAQ at 10 microM had no effect on parathyroid hormone stimulated Ca release. These results suggest that HAQ inhibition of bone resorption appears to be primarily mediated by inhibition of PG biosynthesis.

24-Oxo and 26,23-lactone metabolites of 1,25-dihydroxyvitamin D3 have direct bone-resorbing activity.

The biological activities of several 24-oxo and 26,23-lactone metabolites of vitamin D were determined in bone organ cultures. The 24-oxo metabolites were significantly more potent bone-resorbing agents than the lactones. 1,25-(OH)2-24-oxo-D3 had 0.18 X the bone-resorbing activity of 1,25-(OH)2D3 in fetal rat limb bones and was equipotent with 1,25-(OH)2D3 in neonatal mouse calvaria. In the limb bone system, 1,23,25-(OH)3-24-oxo-D3 had 0.08 X the activity of 1,25-(OH)2D3. 1,25-(OH)2D3 and 1,25-(OH)2-24-oxo-D3 had a similar time course of bone-resorbing effects in both bone culture systems. The most potent of the lactones, 1,25S-(OH)2D3-26,23R-lactone, had approximately 0.009 X the activity of 1,25-(OH)2D3 and approximately 500 times the activity of the 25S-OH-D3-26,23R-lactone. The 25S and 1,25S lactones were more potent than the 25R and 1,25R isomers. In experiments designed to determine whether either 1,25-(OH)2-24-oxo-D3 or 25R-OH-D3-26,23S-lactone could prevent the bone-resorbing activity of 1,25-(OH)2D3, no inhibitory effects were observed. The results suggest that conversion to the lactones represents a substantial inactivation step, whereas conversion to 24-oxo-derivatives results in less reduction in biological activity.

Ethanol effects on harmaline-induced tremor and increase of cerebellar cyclic GMP.

The spectra of pharmacological effects of ethanol and the benzodiazepine show a degree of overlap. Neurophysiological and neurochemical evidence indicates that both ethanol and benzodiazepines facilitate inhibitory neurotransmission mediated by GABA. Diazepam has been reported to inhibit both the tremor and mechanism of cerebellar cyclic GMP caused by harmaline by a neurotransmission in the cerebellum. Because of the similarities between ethanol and benzodiazepines, the effects of ethanol on harmaline-induced tremor and increase of cerebellar cyclic GMP were studied. Ethanol inhibited harmaline-induced tremor at doses as low as 0.1 g/kg. At this low dose, however, a dissociation between inhibition of harmaline tremor and inhibition of the harmaline-induced increase of cerebellar cyclic GMP was observed.

Serial determination of plasma ethanol concentrations in mice.

A technique permitting the serial determination of plasma ethanol concentration (PEC) in mice is described. Samples of blood, 5 microliters in volume, are drawn from the infraorbital plexus. It was demonstrated that this source reflects the rapid dynamics of absorption and clearance after an intraperitoneal injection of ethanol. Samples taken from the tail showed a greatly delayed response and required 30 min to equilibrate with samples taken from the infraorbital plexus. The concentrations of ethanol were determined by gas chromatography. A precolumn removed the nonvolatile constituents thus permitting the injection of plasma directly into the instrument. The ratio of ethanol concentrations in red blood cells to plasma was found to be 0.62. This permitted the estimation of concentration of ethanol in whole blood: (1-0.38 H) X PEC; where H = the hematocrit.

Elevated concentrations of ethanol in plasma do not suppress voluntary ethanol consumption in C57BL mice.

The 24-hr patterns of ethanol intake and resulting concentrations of ethanol in plasma are described for male C57BL/6J mice given free access to water and a 10% v/v solution of ethanol. Animals treated with the alcohol dehydrogenase inhibitor 4-methylpyrazole developed peak plasma concentrations of 116 +/- 20 mg/100 ml, while controls given daily injections of saline exhibited peak plasma concentrations of 11 +/- 7 mg/100 ml. Ethanol consumption as measured by total daily intake and preference was not significantly different in the two groups of mice. The absence of an effect on ethanol consumption despite a tenfold difference in peak plasma levels suggests that concentrations of circulating ethanol within the range observed do not limit voluntary consumption of ethanol.

Development and use of a nonrestraining waveguide chamber for rapid microwave radiation killing of the mouse and neonate rat.

The use of microwave energy for rapid killing of small rodents such as the mouse or rat has become a standard pharmacologic technique since approximately 1975. This method allows investigation of rapidly modulated neurochemical indices, neuromodulatory substances, and some neurotransmitters to be determined at basal concentrations in brain regions and microregions. Previously described devices for use with microwave generators have relied on total body restraining holders in order to properly position rodents and neonates within a closed waveguide during microwave energy exposures. The present information describes two alternate chamber designs which do not require restraint of the rodent. A positioning device is described which must be used with the waveguide chambers. The animal chambers are designed to be used with 2450 MHz energy.