Evaluation of the effects of chronic occupational exposure to metallic mercury on the thyroid parenchyma and hormonal function.

INTRODUCTION: Experiments in animals exposed to mercury (Hg) in different chemical states have shown thyroid parenchymal and hormone alterations. However, these experiments did not allow the establishment of dose-response curves or provide an understanding of whether these Hg effects on the thyroid parenchyma occur in humans. OBJECTIVE: To evaluate the association between chronic occupational exposure to metallic Hg and alterations in thyroid hormones and gland parenchyma 14 years after the last exposure. METHODS: A cross-sectional study including 55 males exposed in the past to metallic Hg and 55 non-exposed males, paired by age, was conducted in the Hospital das Clínicas (Brazil) from 2016 to 2017. Serum concentrations of total and free triiodothyronine (TT3 and FT3), free thyroxine (FT4), thyrotropin (TSH), reverse T3 (RT3), selenium and antithyroid antibody titers were obtained. The Hg and iodine concentrations were measured in urine. The thyroid parenchyma was evaluated by B-mode ultrasonography with Doppler. The nodules with aspects suspicious for malignancy were submitted to aspiration puncture with a thin needle, and the cytology assessment was classified by the Bethesda system. The t test or Mann-Whitney test, Chi-square test and Spearman correlation were used to compare the exposed and non-exposed groups and examine the relationships between the variables. Univariate and multivariate logistic regression models were used to trace determinants of the risk of thyroid hormone alteration. Statistical significance was defined by p < 0.05. RESULTS: The urinary Hg average was significantly higher in the exposed group than in the non-exposed group (p < 0.01). The mean TSH serum concentration in the exposed group was higher, with a statistically significant difference between the groups (p = 0.03). Serum concentrations of TSH exceeded the normality limit (4.20 µIU/ml) in 13 exposed individuals (27.3%) and 4 non-exposed individuals (7.3%), with a statistically significant association between the hormonal increase and exposure to Hg (p = 0.02). In the logistic regression model, exposure to Hg (yes or no) showed an odds ratio = 4.86 associated with an increase of TSH above the normal limit (p = 0.04). The serum concentrations of RT3 showed a statistically borderline difference between the groups (p = 0.06). There was no statistically significant difference between the mean TT3, FT3 and FT4 serum concentrations in the Hg-exposed group compared to the non-exposed group. The proportions of the echogenicity alterations were higher in the exposed group compared to the non-exposed group (27.3% versus 9.1%; p = 0.03). Papillary carcinomas were documented in three exposed individuals and one non-exposed individual. A follicular carcinoma was recorded in one non-exposed individual. CONCLUSIONS: Due to the higher serum TSH concentration and the prevalence of parenchymal alterations in the Hg-exposed group, even after cessation of exposure, it is recommended that the thyroid status of exposed workers be followed for a long period.

Refining hormonal diagnosis of type II 3beta-hydroxysteroid dehydrogenase deficiency in patients with premature pubarche and hirsutism based on HSD3B2 genotyping.

Congenital adrenal hyperplasia due to 3beta-hydroxysteroid dehydrogenase/Delta(5)-Delta(4)-isomerase (3betaHSD), a rare autosomal recessive disorder that affects both sexes, has a heterogeneous clinical presentation ranging from the severe salt-wasting to the non-salt-wasting forms and results from mutations in the HSD3B2 gene. The hormonal criteria for diagnosing the mild variant of 3betaHSD deficiency have been controversial because the initial studies were not based on genetic evidence. We investigated the relationship between the hormonal phenotype and HSD3B2 genotype in 22 patients with clinical and/or biochemical features suggestive of 3betaHSD2 deficiency, including nine female children with premature pubarche, 12 hirsute females, and one boy with salt-wasting and ambiguous genitalia. Serum 17-hydroxypregnenolone (Delta5-17P), cortisol (F), 17-hydroxyprogesterone, dehydroepiandrosterone, and androstenedione levels were determined by RIA and were compared with Tanner pubic hair stage-matched control groups. The genomic DNA was extracted, and the entire HSD3B2 gene was amplified by PCR followed by automatic sequencing. Besides two different mutations previously observed in three patients (T259M and G129R/P222Q mutations), we observed the P222Q mutation in the male patient with salt-wasting form of 3betaHSD2 deficiency. Basal and ACTH-stimulated Delta5-17P levels (nanomoles per liter) ranged from 4-41 (-0.2 to 14 sd) and 36-97 (3.5-15.5 sd), respectively, in patients without mutation in HSD3B2 and from 69-153 (25-57 sd) and 201-351 (36-65 sd), respectively, in patients with mutation in HSD3B2. Basal and ACTH-stimulated Delta5-17P to F ratios ranged from 11-159 (0.5-25 sd) and 42-122 (2.4-11.3 sd), respectively, in patients without mutation in HSD3B2 and from 181-1700 (29-282 sd) and 487-1523 (52-167 sd), respectively, in patients with mutation in HSD3B2. The hormone findings in the genotype-proven patients suggest that the following hormonal criteria are compatible with 3betaHSD2 deficiency in children with premature pubarche: ACTH-stimulated Delta5-17P and Delta5-17P to F ratios at or greater than 201 and 487 nmol/liter, respectively, equivalent to or greater than 36 and 52 sd above matched control mean. Basal and ACTH-stimulated Delta5-17P and Delta5-17P to F ratios in all genotype-proven patients in childhood were unequivocally higher than the levels of either genotype-normal patients. All the other parameters overlapped between the patients with and without mutations in the HSD3B2 gene. In conclusion, genotyping more patients in the present study, we confirm that patients with mutations in the HSD3B2 gene have extremely elevated basal and ACTH-stimulated Delta5-17P levels and Delta5-17P to F ratios. Therefore, these data refine the hormonal criteria proposed to predict more accurately 3betaHSD2 deficiency.

Combined pituitary hormone deficiency caused by a novel mutation of a highly conserved residue (F88S) in the homeodomain of PROP-1.

Mutations in the pituitary-specific paired-like homeodomain transcription factor, PROP-1, result in combined pituitary hormone deficiency. We studied a Brazilian girl, offspring of first cousins, who presented with short stature and deficiencies of GH, TSH, PRL, LH, and FSH. Her cortisol response to hypoglycemia was determined at age 4.9, 10.7, and 14.1 yr and remained normal. Magnetic resonance imaging at the age of 9 yr revealed an anterior pituitary lobe of diminished height (3 mm; normal, 4.5 +/- 0.6), but radiography revealed a sella turcica volume above the normal mean. Direct sequencing of the PROP-1 gene revealed homozygosity for a novel 263T>C transition that results in the replacement of a highly conserved phenylalanine by serine at codon 88 (F88S). F88 constitutes the hydrophobic core of the first helix of the homeodomain of PROP-1, and the substitution by the polar residue serine is expected to alter the secondary structure and impair binding of the mutated PROP-1 to DNA target sequences. The F88S mutation (which corresponds to murine F85S) was introduced into the murine Prop-1 complementary DNA and its consequences on DNA binding and trans-activation were assessed in vitro. In contrast to wild-type Prop-1, the F88S mutant showed no significant DNA binding to a PRDQ9 Prop-1 response element in gel shift assays. Transcriptional activation of a luciferase reporter gene containing a PRDQ9 site upstream of a simian virus 40 promoter was reduced to approximately 34% compared with that of wild-type Prop-1 in transiently transfected TSA-201 human embryonic kidney cells. The F88S mutation further expands the repertoire of mutations in PROP-1.

Origin of an ovarian steroid cell tumor causing isosexual pseudoprecocious puberty demonstrated by the expression of adrenal steroidogenic enzymes and adrenocorticotropin receptor.

Ovarian steroid cell tumors are rare neoplasms composed of typical steroid hormone-secreting cells. Most ovarian steroid cell tumors, however, cannot be appropriately classified on a morphological basis, because the neoplastic cells closely resemble adrenal cortical cells. Nevertheless, the true adrenal origin of such tumors has been difficult to demonstrate. Here we report a 3-yr-old girl with isosexual pseudoprecocious puberty due to an ovarian steroid tumor whose adrenal cell origin was determined by the presence of messenger ribonucleic acid (mRNA) of adrenal-specific steroidogenic P450 enzymes (P450c11 and P450c21) and ACTH receptor (ACTHR). Her height was +2.3 SD, and she had Tanner stage III breast development, Tanner stage II pubic hair, and a normal clitoris. Bone age was 5 yr. Basal gonadotropin levels were undetectable (<0.6 U/L for LH and <1.0 U/L for FSH) and remained undetectable after stimulation with 100 microg GnRH, i.v. Basal serum testosterone and 17-hydroxyprogesterone levels were slightly elevated, whereas basal serum androstenedione, estradiol, and dehydroepiandrosterone sulfate levels were clearly elevated. Pelvic ultrasound disclosed an enlarged uterus and an adnexal multicystic mass in the right ovary, and pathological studies disclosed an ovarian steroid cell tumor. To establish the cellular origin of the tumor we determined the presence of mRNA for P450c11, P450c21, and ACTHR in tumor tissue and normal adrenal and ovarian tissue. Detection of ACTHR, P450c21, and P450c11 mRNAs isoforms was achieved in tumoral and adrenal control tissue, but not in the ovary control tissue. The RT-PCR products of P450c11 from adrenal control tissue were composed by both BglI-sensitive and -resistant complementary DNAs, indicating the presence of both P450c11AS and P450c11beta, whereas RT-PCR product from the tumor was resistant to BglI digestion, indicating only the presence of P450c11beta. We conclude that the histological origin of so-called adrenal rest tumor could be reliably determined by assessing the expression of specific genes in the tumor as P450c11beta and P450c21. The use ofthese molecular tools will allow a more precise classification of an important subset of the ovarian steroid cell tumors and can help to identify ectopic adrenal tissue in ovary and testis.

Apparent mineralocorticoid excess in a Brazilian kindred: hypertension in the heterozygote state.

BACKGROUND: Apparent mineralocorticoid excess (AME) is a cause of low-renin, low-aldosterone hypertension in which cortisol acts as a mineralocorticoid. The condition reflects an inability to inactivate cortisol to cortisone due to defective activity of the type 2 isozyme of 11beta-hydroxysteroid dehydrogenase (11beta-HSD2). Homozygous mutations in 11beta-HSD2 gene in patients with AME have been described. A 7-year-old Brazilian girl had previously been found to have AME. Her father recently presented with mineralocorticoid hypertension at age 38 years. OBJECTIVE: To describe the clinical details, to perform steroid analyses and to assess the molecular basis for the hypertension in this kindred. METHODS: The 11beta-HSD2 gene was amplified from genomic DNA by the polymerase chain reaction and sequenced by direct chain-termination sequencing on an automatic DNA sequencer. The sequencing results were validated by restriction-site polymorphism. The mutant 11beta-HSD2 protein was expressed in Chinese hamster ovary polyoma cells and enzymatic activity was assessed by metabolizing cortisol in vitro. RESULTS: Sequence analysis of genomic DNA revealed a novel C1061T point mutation in exon V of the human 11beta-HSD2 gene, resulting in an amino acid substitution of alanine by valine at codon 328 of the enzyme protein (A328V). Expression studies confirmed that the mutant protein was devoid of 11beta-HSD2 activity. A HhaI restriction-site polymorphism confirmed that the proband was homozygous for the mutation whereas both parents were heterozygotes. The father of the proband had hypertension, a normal serum potassium level, suppressed plasma renin activity and plasma aldosterone level and a moderately elevated urinary cortisol: cortisone metabolite ratio. CONCLUSIONS: AME in this kindred is caused by a novel mutation in the 11beta-HSD2 gene. Detection of hypokalaemia, at least in this kindred, is an insensitive screening test for mineralocorticoid-based hypertension. In contrast to results from previously investigated kindreds, we have demonstrated that this kindred has an abnormal phenotype in the heterozygote state. Further studies are now required in order to evaluate the role of 11beta-HSD2 activity in the pathophysiology of 'essential' hypertension.

EM574, an erythromycin derivative, improves delayed gastric emptying of semi-solid meals in conscious dogs.

The gastroprokinetic effects of de(N-methyl)-N-isopropyl-8, 9-anhydroerythromycin A 6,9-hemiacetal (EM574), a non-peptide motilin receptor agonist, were investigated in conscious dogs in a normal state and with experimentally-induced gastroparesis. Gastric emptying of semi-solid meals was assessed indirectly from acetaminophen absorption with simultaneous recording of gastric antral motility. In the normal state, post-prandial intraduodenal administration of EM574 (0.03 mg/kg) [corrected] stimulated antral motility and significantly enhanced gastric emptying as potently as did intravenous porcine motilin (0.003 mg/kg/h). Intraduodenal cisapride at 1 mg/kg denal cisapride at 1 mg/kg elicited antral contractions and tended to accelerate gastric emptying but at 3 mg/kg, gastric emptying was not enhanced despite a further increase in the motor index. In dogs with gastroparesis induced by intraduodenal oleic acid or intravenous dopamine, EM574 (0.03 mg/kg) increased antral motility and reversed the delayed gastric emptying completely. Cisapride (1 mg/kg) partially ameliorated the impaired emptying under these conditions. In atropinized dogs, no acceleration of gastric emptying by EM574 was observed. These results indicate that EM574 potently accelerates gastric emptying of caloric meals in dogs in a normal state and with experimentally-induced gastroparesis, and also suggest that the effect is mediated through stimulation of a cholinergic neural pathway.

EM574, an erythromycin derivative, is a motilin receptor agonist in the rabbit.

This study was performed to examine whether an erythromycin derivative, de(N-methyl)-N-isopropyl-8,9-anhydroerythromycin A 6,9-hemiacetal (EM574) is a motilin receptor agonist in the rabbit gastrointestinal tract. EM574 and porcine motilin induced contractions in segments of isolated rabbit intestine with pEC50 values of 8.26 +/- 0.04 and 8.69 +/- 0.07, respectively, but not in rat or guinea pig preparations. The sensitivity and efficacy of the response to both compounds in rabbits decreased aborally and was insensitive to pretreatment with atropine or tetrodotoxin, but was markedly suppressed under Ca(2+)-free conditions. EM574 and porcine motilin specifically displaced [125I-Tyr23]canine motilin bound to gastric antral smooth muscle homogenates with plC50 values of 8.21 +/- 0.13 and 9.20 +/- 0.11, respectively. The pEC50 value for the contractile response and plC50 value for the receptor binding for motilin, EM574, erythromycin A and three other derivatives correlated well (r = 0.94, P < 0.01). Tissue section autoradiography in the antrum revealed that specific labeled motilin binding sites were localized in the circular muscle layer and myenteric plexus, and could be reduced in the presence of an excess of EM574. These results indicate that EM574 is a potent motilin receptor agonist in the rabbit gastrointestinal tract.

Genotyping of the type II 3beta-hydroxysteroid dehydrogenase gene (HSD3B2) in women with hirsutism and elevated ACTH-stimulated delta(5)-steroids.

OBJECTIVE: Genotyping of the HSD3B2 gene in females with hirsutism and elevated ACTH-stimulated Delta(5)-steroids. DESIGN: Fourteen adult females whose ACTH-stimulated 17-hydroxypregnenolone (17OH-PREG) levels were elevated (>/= 2.3 SD). SETTING: University hospital outpatient clinic and molecular endocrinology laboratory. PATIENT(S): Thirteen women with hirsutism and one with virilization. INTERVENTION(S): ACTH-stimulation test and genotyping of the HSD3B2 gene. MAIN OUTCOME MEASURE(S): The four exons and exon-intron boundaries of the HSD3B2 gene were amplified by the use of polymerase chain reaction and were screened for mutations by denaturing gradient gel electrophoresis. The fragments that were found to have abnormal migration on denaturing gradient gel electrophoresis were directly sequenced. RESULT(S): No mutations were found in 13 patients who had mild to moderate elevations in ACTH-stimulated 17OH-PREG levels, and the T259M mutation was identified in the woman with virilization and extremely high 17OH-PREG levels. CONCLUSION(S): Mutations in the HSD3B2 gene were not found in women with hirsutism and mild-to-moderate elevations in ACTH-stimulated 17OH-PREG levels.

TAK-147, an acetylcholinesterase inhibitor, increases choline acetyltransferase activity in cultured rat septal cholinergic neurons.

TAK-147, a potent acetylcholinesterase (AChE) inhibitor, potentiated choline acetyltransferase (ChAT) activity in cultured rat septal cholinergic neurons in a concentration-dependent manner with an EC50 value of 4.47 nM. Donepezil, another potent AChE inhibitor, also increased ChAT activity although its potency was less than that of TAK-147. Other AChE inhibitors (rivastigmine, tacrine, physostigmine and neostigmine) showed no effect. The effects of TAK-147 were greater in the presence of NGF, suggesting a synergistic action of TAK-147 and NGF. TAK-147 and donepezil showed high affinity for sigma receptors, whereas tacrine and physostigmine did not. Haloperidol and ifenprodil, high-affinity sigma ligands, potently enhanced ChAT activity in the septal neurons. These results suggest that TAK-147 may have neurotrophic activity on central cholinergic neurons, not via AChE inhibition but possibly via an effect on tau receptors.

Mutagenicity of N-acetyl and N,N'-diacetyl derivatives of 3 aromatic amines used as epoxy-resin hardeners.

3 epoxy-resin hardeners, 4,4'-diaminodiphenyl ether (DDE), 4,4'-diaminodiphenylmethane (DDM), and 4,4'-diaminodiphenylsulfone (DDS), and their N-acetyl and N,N'-diacetyl derivatives were examined for their mutagenicity using Salmonella typhimurium TA98 and TA100 as the tester stains and an S9 mix containing a rat-liver 9000 X g supernatant fraction as the metabolic activation system. DDE and DDM were mutagenic towards TA98 and TA100 in the presence of S9 mix while DDS exhibited no significant mutagenic activity towards these tester strains. These epoxy-resin hardeners were metabolized in vivo and their N-acetyl and N,N'-diacetyl metabolites were found in the urine. Among these acetyl metabolites, only N-acetyl-DDE was found to be mutagenic towards TA98 and TA100 in the presence of S9 mix. None of these acetyl metabolites exhibited significant mutagenic activity towards these tester strains in the absence of S9 mix.

Chemical modification of fumagillin. II. 6-Amino-6-deoxyfumagillol and its derivatives.

6-Amino-6-deoxyfumagillol (5) was synthesized by reductive amination of 6-oxo-6-deoxyfumagillol (4), which was obtained by oxidation of fumagillol (2). The reduction proceeded stereoselectively by the equatorial attack of hydride and 5 was found to have the same stereochemistry as that of 2. Several derivatives of 5 were prepared and most of them showed anti-angiogenic activity comparable to that of fumagillol derivatives.

Potent anti-angiogenic action of AGM-1470: comparison to the fumagillin parent.

The anti-angiogenic activity of AGM-1470, a new synthetic analog of fumagillin isolated from Aspergillus fumigatus, was extensively examined both in vitro and in vivo using four different types of assay and compared to that of the fumagillin parent. Locally administered AGM-1470 inhibited the angiogenesis in the chick embryo chorioallantoic membrane assay and the rat corneal assay. In the rat sponge implantation assay, systemically administered AGM-1470 inhibited angiogenesis induced by basic fibroblast growth factor. Furthermore, in the rat blood vessel organ culture assay, AGM-1470 (1-1,000 ng/ml) was found to selectively inhibit the capillary-like tube formation of endothelial cells with a minimal effect on the non-endothelial cell growth. AGM-1470 showed more potent anti-angiogenic activity and less toxicity than the fumagillin parent. Therefore, AGM-1470 is much better than the fumagillin parent as anti-angiogenic compound.

A novel immunosuppressant, IR-1116, which has a different biological mechanism from that of cyclosporine A.

The effects of an epifumagillol derivative, IR-1116, on human lymphocytes in vitro, mouse antibody production in vivo and rat renal and cardiac transplantation were investigated. IR-1116 suppressed the human primary mixed lymphocyte reaction (MLR) in a dose-dependent manner with a value of the dose required for 50% suppression of 0.32 microM. IR-1116 suppressed the antibody production by human B lymphocytes activated with pokeweed mitogen or Epstein-Barr virus, but did not affect the interleukin-2 production by human lymphocytes stimulated with phytohemagglutinin. Furthermore, IR-1116 did not have any effect on the proliferation of B or T lymphocytes. IR-1116 is thought to have a different mechanism from cyclosporine A (CsA). BALB/c mice were immunized by s.c. injection of bovine gamma-globulin (BGG) and then received IR-1116 or CsA by i.p. injection at a dose of 20 mg/kg every other day for 2 weeks. IR-1116 suppressed the production of serum antibodies against BGG as strongly as CsA. In this experiment, IR-1116 apparently showed no adverse effects, while CsA-treated mice suffered from diarrhea and appeared to be irritated. Histological studies showed that IR-1116 suppressed the formation of germinal centers in the spleen of immunized mice. Flow cytometric analysis showed that IR-1116 caused a reduction of B lymphocyte population in splenocytes. On the other hand, CsA caused a marked reduction of helper T lymphocyte population in splenocytes and thymocytes. Furthermore, the i.p. administration of IR-1116 prolonged the survival time in a rat renal allograft model and the vital period of grafted hearts in rats. Based on the above, IR-1116 seems to be a new type of immunosuppressant acting via novel mechanisms different from those of immunosuppressants such as CsA, a potent T lymphocyte suppressant.

Motilides, macrolides with gastrointestinal motor stimulating activity. I. O-substituted and tertiary N-substituted derivatives of 8,9-anhydroerythromycin A 6,9-hemiacetal.

Chemical modifications of 8,9-anhydroerythromycin A 6,9-hemiacetal (1), which showed gastrointestinal motor stimulating (GMS) activity 10 times more potent than that of erythromycin A (EM-A), were undertaken to search for derivatives having stronger GMS activity and no antimicrobial activity; details are described in this and a subsequent paper. Displacement of a methyl group of the dimethylamino group of 1 with an ethyl group and an isopropyl group provided de(N-methyl-N-ethyl-8,9-anhydroerythromycin A 6,9-hemiacetal (55) and de(N-methyl)-N-isopropyl-8,9-anhydroerythromycin A 6,9-hemiacetal (58), respectively. They showed significant GMS activity and no antibacterial activity. In particular, the GMS activity of 58 was increased to 248 times that of EM-A. EM-A and the derivatives obtained in this study mimic exogenous motilin in the dog. The name "motilide", meaning a motilin-like macrolide, is proposed for this new family of macrolide compounds.