Synthesis and complement inhibitory activity of B/C/D-ring analogues of the fungal metabolite 6,7-diformyl-3',4',4a',5',6',7',8',8a'-octahydro-4,6',7'-trihydroxy- 2',5',5',8a'-tetramethylspiro[1'(2'H)-naphthalene-2(3H)-benzofuran

This paper reports the synthesis and the bioassay of 4-methoxy- and 4-hydroxyspiro[benzofuran-2(3H)-cyclohexane] partial analogues (5) of the complement inhibitory sesquiterpene fungal metabolite 6,7-diformyl-3',4',4a',5',6',7',8',8a'-octahydro-4,6',7'-trihydroxy-2',5',5',8a'-tetramethylspiro[1'(2'H)-naphthalene-2(3H)-benzofuran] (1a, K-76) and its silver oxide oxidized product (1b, K-76COOH). The described target compounds represent spirobenzofuran B/C/D-ring analogues lacking the A-ring component of the prototype structure. The target compounds were evaluated by the inhibition of total hemolytic complement activity in human serum. It was observed that the structurally simplified analogue 4-methoxyspiro[benzofuran-2(3H)-cyclohexane]-6-carboxylic acid (5a) exhibited an IC(50) = 0.53 mM similar to the IC(50) = 0.57 mM that was observed for the natural product derivative 1b. Exhibiting an IC(50) = 0.16 mM, the three-ringed partial structure 6-carboxy-7-formyl-4-methoxyspiro[benzofuran-2(3H)-cyclohexane] (5k)was found to be the most potent target compound. Like the natural product, 5k appears to inhibit primarily at the C5 activation step and inhibits both the classical and alternative human complement pathways. Several other analogues inhibited complement activation in vitro at concentrations similar to those required for inhibition by the natural product 1b.     

Biomonitoring equivalents for 2,2',4,4',5-pentabromodiphenylether (PBDE-99)

Biomonitoring Equivalents (BEs) are defined as the concentration or range of concentrations of a chemical or its metabolite in a biological medium (blood, urine, or other medium) that is consistent with an existing health-based exposure guideline such as a reference dose (RfD) or tolerable daily intake (TDI). BE values can be used as a screening tool for the evaluation of population-based biomonitoring data in the context of existing risk assessments. This study reviews health based risk assessments and exposure guidance values for 2,2',4,4',5-pentabromodiphenylether (PBDE-99) from Health Canada and the United States Environmental Protection Agency (US EPA). Toxicokinetic data from laboratory animals and humans are reviewed. A BE value corresponding to the US EPA RfD is derived here for PBDE-99 based on the assumption of chronic steady-state exposure, distribution into body lipids, and a previously-estimated first-order half-life of elimination of 1040days. The steady-state lipid-adjusted BE(RfD) is 520ng/g lipid. Sources of uncertainty relating to the underlying toxicokinetic and toxicologic database for PBDE-99 and the simultaneous exposure to multiple PBDE congeners are discussed. The BE(RfD) value may be used as a screening tool for evaluation of population biomonitoring data for PBDE-99 in the context of the existing US EPA risk assessment and can assist in prioritization of the potential need for additional risk assessment efforts for PBDE-99 relative to other chemicals.     

Pyrazino(1,2-a)- and 1,4-diazepino(1,2-a)- indoles. III. Synthesis of pyrrolo(2',1':3,4)pyrazino(1,2-a)indoles and pyrrolo(2',1':3,4)-1,4-diazepino(1,2-a) indoles; isoindolo(2',1':3,4)pyrazino(1,2-a)-indoles and isoindolo(2',1':3,4)-1,4-diazepino(1,2-a)indoles; pyrazino(3,2,1-jk)- carbazoles and 1,4-diazepino(3,2,1-jk) carbazoles]

Pyrrolo(2',1':3,4)pyrazino (or 1,4-diazepino) (1,2-a)indoles and isoindolo(2',1':3,4)pyrazino (or 1,4-diazepino) (1,2-a)indoles were synthesized by reaction of 1-(2-aminoethyl)indole or 1-(3-aminopropyl)indole with gamma-keto and o-acylbenzoic acids. In addition, a novel synthetic route for the preparation of pyrazino(3,2,1-jk)- and 1,4-diazepino(3,2,1-jk)carbazoles has been realized.     

Effects of oral erythrosine (2',4',5',7'-tetraiodofluorescein) on the pituitary-thyroid axis in rats

Erythrosine (FD&C Red Dye No.3) is a tetraiodinated derivative of fluorescein. Rats fed a 4% erythrosine diet for 30 months beginning in utero have an increased incidence of thyroid adenomas and adenocarcinomas. These tumors may be secondary to increased stimulation of the thyroid gland by TSH. This study was undertaken to determine if dietary erythrosine disrupts the pituitary-thyroid axis thereby altering serum thyroid hormone levels. TSH levels, or the pituitary's response to TRH. Rats were fed diets containing erythrosine (0.5, 1.0, 4.0%), sodium iodide (0.16%), or fluorescein (1.6%) for 3 weeks after which TRH testing was performed in vivo. Erythrosine produced a dose-dependent increase in serum T4 levels. With the 4% erythrosine diet, serum T4 and T3 levels and the free-T4 index were significantly increased, whereas the free-T3 index were significantly increased, whereas the free-T3 index was unchanged. Rats fed the 4.0% erythrosine diet had an exaggerated TSH response to TRH; 10 min after the TRH injection, serum TSH levels were 80% greater than TSH levels of control rats. Short-term administration of erythrosine to rats decreased hepatic T3 production by decreasing its conversion of T4 to T3, indicating that erythrosine decreases hepatic 5'-deiodinase activity. These data demonstrate that dietary ingestion of 4% erythrosine disrupts the pituitary-thyroid axis as evidenced by an increased TSH response to TRH. This effect is mediated by erythrosine or an iodinated metabolite, since ingestion of its fluorescein nucleus had no effect. Erythrosine's effects were not likely mediated by iodide, because serum T4 and T3 levels were elevated and iodide administration did not increase the TSH response to TRH. These data suggest that erythrosine increases the pituitary's TSH response to TRH by altering thyrotroph cell conversion of T4 to T3. Chronic erythrosine ingestion may promote thyroid tumor formation in rats via chronic stimulation of the thyroid by TSH.     

Toxicity of PCB 77 (3,3',4,4'-Tetrachlorobiphenyl) and PCB 118 (2,3',4,4',5-Pentachlorobiphenyl) in the Rat Following Subchronic Dietary Exposure

The toxicity of 3,3',4,4'-tetrachlorobiphenyl (PCB 77) and 2,3',4,4',5-pentachlorobiphenyl(PCB 118) was investigated in rats following subchronic dietaryexposure. Groups of 10 male and 10 female weanling Sprague-Dawleyrats were administered PCB 77 In the diet at 0, 10, 100, 1000,or 10,000 ppb for 13 weeks. PCB 118 was administered to malesin the diet at 0, 10, 100, 1000, and 10,000 ppb, while the femalegroups received 0, 2, 20, 200, or 2000 ppb of the congener for13 weeks. Growth rate and food consumption were not affectedby treatment. No clinical signs of toxicity were observed. Increasedspleen weight occurred in male rats fed 1000 or 10,000 ppb PCB77. Male rats receiving 10,000 ppb PCB 118 had increased liverweight and hepatic ethoxyresorufin O-deethylase (EROD) activity.Increased hepatic EROD activity but not liver weight was observedin female rats given the 2000-ppb PCB 118 diet. Increased ERODactivity was also noted in male rats given 10,000 ppb and infemale groups receiving 1000 or 10,000 ppb PCB 77. Male ratsexposed to 10,000 ppb PCB 77 had decreased vitamin A in theliver and lung and elevated levels in the kidney. Liver vitaminA of both 1000- and 10,000-ppb PCB 77 female groups was decreased.PCB 118 had no effects on tissue vitamin A at the levels studied.No hematological changes or serum biochemical changes were seenin any of PCB 118- and PCB 77-treated groups, nor were liveruroporphyrin levels altered. A reduction in dopamlne and homovanillinicacid in the substantia nigra region of the brain was observedin female rats fed 2000 ppb PCB 118, while 10,000 ppb PCB 77was associated with an elevation in 3,4-dihydroxyphenylaceticacid in the nucleus accumbens region of male rat brains. Mildto moderate changes were observed in the liver and thyroid ofrats given PCB 77 or PCB 118. PCB 118 accumulated in a dose-dependentmanner in fat and to a much lesser extent in liver. In contrast,very low levels of PCB 77 residue were found in the tissuesexamined. Based on the above data It was concluded that theNOAEL of PCB 77 is 100 ppb in diet or 8.7 sglkg and that ofPCB 118 is 200 ppb in diet or 17 µg/kg body wt/day.     

Effects of oral erythrosine (2',4',5',7'-tetraiodofluorescein) on thyroid function in normal men

Erythrosine (Er), a tetraiodinated derivative of fluorescein, is a coloring agent widely used in foods, cosmetics, and pharmaceutical products. Because of its high iodine content and previous reports demonstrating an inhibitory effect of erythrosine on hepatic 5'-monodeiodination, we studied the effects of this compound on thyroid function and serum and urinary iodide concentrations in normal subjects. Thirty normal men, equally divided into three treatment groups, each received a 14-day course of oral Er in doses of 20, 60, or 200 mg/day. Serum thyroxine (T4), triiodothyronine (T3), reverse T3 (rT3), thyroid stimulating hormone (TSH), protein-bound iodide (PBI), and total iodide concentrations, serum T3-charcoal uptake, and 24-hour urinary iodide excretion were measured on Days 1, 8, and 15. Thyrotropin-releasing hormone (TRH) tests were performed on Days 1 and 15. There were no significant changes in serum T4, T3, rT3, and T3-charcoal uptake values at any dose. In men receiving 200 mg Er/day, the mean basal serum TSH concentration increased significantly from 1.7 +/- 0.1 (SE) on Day 1 to 2.2 +/- 0.1 microU/ml on Day 15 (p less than 0.05), and the mean peak TSH increment after TRH increased from 6.3 +/- 0.5 to 10.5 +/- 1.0 microU/ml (p less than 0.05). There were no significant changes in basal or peak TSH responses in the men receiving 20 or 60 mg Er/day. Significant dose-related increases in serum total iodide and PBI concentrations occurred during all three doses, and significant dose-related increases in urinary iodide excretion occurred during the 60 and 200 mg/day Er doses. These data suggest that the increase in TSH secretion induced by Er was related to the antithyroid effect of increased serum iodide concentrations, rather than a direct effect of Er on thyroid hormone secretion or peripheral metabolism.     

Developmental Toxicity of PCB 126 (3,3',4,4',5-Pentachlorobiphenyl) in Nestling American Kestrels (Falco sparverius)

Planar PCB congeners are embryotoxic and teratogenic to birdsincluding American kestrels. The developmental toxicity of 3,3',4,4',5-pentachlorobiphenyl(PCB 126) was studied in the posthatching kestrel as a modelfor the eagle. Nestlings were dosed orally for 10 days with5 µl/g body weight of corn oil (controls) or the planarPCB 126 at concentrations of 50, 250, or 1000 ng/g body weight.Dosing with 50 ng/g of PCB 126 resulted in a hepatic concentrationof 156 ng/g wet weight, liver enlargement and mild coagulativenecrosis, over 10-fold increases in hepatic microsomal ethoxyresorufin-O-dealkylaseand benzyloxyresorufin-O-dealkylase, and approximately a 5-foldincrease in methoxyresorufin-O-dealkylase. At this dose, mildto moderate lymphoid depletion of the spleen was apparent, aswere decreased follicle size and content of the thyroid. At250 ng/g, concentration of PCB 126 in the liver was 380 ng/gwith increasing multifocal coagulative necrosis, decreased bonegrowth, decreased spleen weight with lymphocyte depletion ofthe spleen and bursa, and degenerative lesions of the thyroid.At 1000 ng/g, the liver concentration was 1098 ng/g, accompaniedby decreased bursa weight, decreased hepatic thiol concentration,and increased plasma enzyme activities (ALT, AST, and LDH-L)in addition to the previous effects. Highly significant positivecorrelations were noted between liver concentrations of PCB126 and the ratio of oxidized to reduced glutathone. These findingsindicate that nestling kestrels are more susceptible to PCB126 toxicity than adults, but less sensitive than embryos, andthat planar PCBs are of potential hazard to nestling birds.     

Cytotoxicity of (2,2':6',2'-terpyridine)platinum(II) complexes to Leishmania donovani, Trypanosoma cruzi, and Trypanosoma brucei

A range of (2,2':6',2'-terpyridine)platinum(II) complexes are shown to possess antiprotozoal activity in vitro against Leishmania donovani, Trypanosoma cruzi, and Trypanosoma brucei,the causative organisms of tropical diseases leishmaniasis and trypanosomiasis. The best compounds caused 100% and 78% inhibition of growth of the intracellular amastigote forms of L. donovani and T. cruzi, respectively, at a concentration of 1 microM and 100% inhibition of growth of the bloodstream trypomastigote forms of T. brucei at a concentration of 0.03 microM. The results obtained with complexes in which the fourth ligand to platinum(II) is capable of being substituted with a substitution inert hydroxyethanethiolate complex are compared. The ammine complexes show high antiprotozoal activity suggesting that the trans influence of the 2,2':6',2'-terpyridine ligand has a profound effect on the ease of displacement of the fourth ligand in (2,2':6',2' -terpyridine)platinum(II) complexes, although nonbonded interaction between the ammine ligand and the 6 and 6' ' hydrogens probably also weakens the ligation to Pt(II).     

Cytotoxicity of 2,2':6',2' '-terpyridineplatinum(II) complexes against human ovarian carcinoma.

2,2':6',2'-Terpyridineplatinum(II) complexes are shown to possess cytotoxicity against a number of human ovarian tumor cell lines. Many of the complexes show similar activity against cisplatin- and doxorubicin-resistant cell lines as the parental cells suggesting that there is little or no cross-resistance with cisplatin or doxorubicin. The cytotoxicity of bis[2,2':6',2'-terpyridineplatinum(II)] complexes is strongly dependent on the nature of the linker. Bis[2,2':6',2'-terpyridineplatinum(II)] complexes with a flexible linker at the 4'-position show poor cytotoxicity; by contrast bis[2,2':6',2'- terpyridineplatinum(II)] complexes with rigid and short linkers at platinum(II) are strikingly effective. Several of the compounds show greater cytotoxicity against human ovarian cell lines than carboplatin, the therapeutic agent currently advocated for the treatment of human ovarian cancers.     

Heme oxygenase 1 mediates anti-inflammatory effects of 2',4',6'-tris(methoxymethoxy) chalcone

We report that the synthetic chalcone 2',4',6'-tris(methoxymethoxy) chalcone (TMMC) is an anti-inflammatory compound that reduces nitric oxide (NO) production by inhibiting of inducible NO synthase (iNOS) expression, and that TMMC decreases the degradation of the inhibitory factor kappaB, leading to inhibition of nuclear factor-kappaB translocation into the nucleus in lipopolysaccharide (LPS)-activated RAW 264.7 macrophages. We also demonstrate that TMMC by itself is a potent inducer of heme oxygenase 1 (HO-1). Inhibition of HO-1 activity or scavenging of carbon monoxide, a byproduct of heme degradation, significantly attenuated this anti-inflammatory action. Treating cells with the specific p42/44 MAPK inhibitor, PD98059, blocked the TMMC-mediated induction of HO-1 and the inhibition of LPS-stimulated expression of iNOS. TMMC also depleted intracellular GSH. Our data suggest that TMMC exerts an anti-inflammatory effect in macrophages through a mechanism that involves the induction of HO-1, which is mediated by activation of p42/44 MAPK and GSH depletion.     

Dietary accumulation and biochemical responses of juvenile rainbow trout (Oncorhynchus mykiss) to 3,3',4,4',5-pentachlorobiphenyl (PCB 126)

Juvenile rainbow trout (Oncorhynchus mykiss) (initial weights 2-5 g) were exposed to three dietary concentrations (0, 12.4 and 126 ng g(-1), wet weight) of a 14C-labelled 3,3',4,4',5-pentachlorobiphenyl (PCB 126) for 30 days followed by 160 days of clean food. We assessed bioaccumulation, histology (liver and thyroid) and biochemical responses (liver ethoxyresorufin-O-deethylase (EROD), liver vitamins (retinoids and tocopherol) and muscle thyroid hormone levels) along with growth and survival. The half-life of PCB 126 in the rainbow trout ranged from 82 to 180 days while biomagnification factors (BMF) ranged from 2.5 to 4.1 providing further evidence that PCB 126 is among the most bioaccumulative PCB congeners. Toluene extractable 14C declined with time in the trout suggesting the possibility of some biotransformation and/or covalent bonding with biological macromolecules. The threshold for liver EROD induction by PCB 126 was approximately 0.1 ng g(-1) (wet weight). EROD activities in the low- and high treatments were 9 and 44 times greater than control, respectively, and remained elevated throughout the experiment. EROD activity was correlated with whole body concentrations of PCB 126 although there was evidence of EROD activity suppression in the highly exposed fish. Liver didehydroretinoids and tocopherol concentrations were depressed by the high PCB 126 dose after 30 days exposure. Initially, muscle concentrations of thyroxine (T4) and triiodo-L-thyronine (T3) declined as the fish grew during the experiment, and exposure to PCB 126 accelerated the growth related decline. More information is needed to assess the functional significance of the reduced muscular stores of thyroid hormones. Despite the changes in liver EROD, liver vitamins and muscle thyroid hormones, liver and thyroid histology in trout examined after 30 days exposure and growth parameters were unaffected by PCB 126. This indicates that the functional competences of the physiological factors associated with growth were maintained under the experimental conditions.     

Synthesis and characterization of oligonucleotides containing 5',8-cyclopurine 2'-deoxyribonucleosides: (5'R)-5',8-cyclo-2'-deoxyadenosine, (5'S)-5',8-cyclo-2'-deoxyguanosine, and (5'R)-5',8-cyclo-2'-deoxyguanosine.

Radiation-induced degradation of purine and pyrimidine nucleosides gave rise to carbon-bridged cyclocompounds. Such cyclonucleosides represent a class of tandem lesions in which modification of both the base and 2-deoxyribose has occurred. A solid-phase synthetic method was designed for the incorporation of both 5'R and 5'S diastereoisomers of 5',8-cyclopurine 2'-deoxyribonucleosides into oligodeoxynucleotides to facilitate the assessment of the biochemical and biophysical features of such lesions. We report the preparation of the phosphoramidite synthons of (5'R)-5', 8-cyclo-2'-deoxyadenosine (2), (5'S)-5',8-cyclo-2'-deoxyguanosine (3), and (5'R)-5',8-cyclo-2'-deoxyguanosine (4). Fully protected compounds 10, 18, and 25 were then inserted into several oligonucleotides by automated procedures. Analysis of modified DNA oligomers 26-31 by electrospray mass spectrometry and enzymatic digestions with exo- and endonucleases confirmed the base compositions and the integrity of free radical-induced tandem lesions 2-4 that were chemically inserted.     

Effects of pure chlorobiphenyls (2,4',5-trichlorobiphenyl and 2,2',4,4',5,5'-hexachlorobiphenyl) on the post-natal growth in mice.

The post-natal growth rate of young mice, pre- and post-natally exposed to either 2,4',5-trichlorobiphenyl or 2,2',4,4',5,5',-hexachlorobiphenyl has been studied. The results show that both the chlorobiphenyls have the ability toincrease the post-natal growth rate in mice and they indicate that this ability of 2,4',5-trichlorobiphenyl is most pronounced in females whereas that of 2,2',4,4',5,5',-hexachlorobiphenyl is most pronounced in males.     

Investigations on the occurrence of cytidine 3',5' monophosphate (cCMP) in tissues

A specific and sensitive radioimmunoassay (RIA) for the detection of cCMP was developed. Antibodies were prepared by immunization of rabbits with succinyl cCMP conjugated to human albumin. In the RIA commercially available 3H-cCMP was used as labeled antigen. Low amounts (0.4-2.6 pmol/g w.wt.) of immunoreactive material could be detected in extracts of brain, heart, ileum, kidney, liver, lung, spleen, testis and uterus from rats, guinea-pig and frogs. The immunoassayable material in rat liver was subjected to column chromatography on AG 1-X8 anion exchange resin. The radioimmunoassayable material isolated from the liver did not cochromatograph with authentic tritiated or unlabeled cCMP. The chromatographic properties of the immunoreactive material did not change in regenerating livers - following partial hepatectomy, or after pronase treatment. However, after treatment with pronase the amount of assayable material, extracted from the rat liver, increased. It is suggested that cCMP - as such - is not present, in detectable amounts, in extracts prepared by commonly used procedures for isolation of cyclic nucleotides. The chemical nature of the cCMP immunoreactive material isolated from rat liver is at present unclear.