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1991 Fluoride Abstracts. Part 1.

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Fluoride 1991; 14(3):85-89

Editorial: National Toxicology Program - Critique of Peer Review Draft Report

RJ Carton

Representing the Professional's Union, Local 2050, National Federation of Federal Empoyees (at the Environmental Protection Agency headquarters), Washington DC 20046. USA.

Excerpt:

... The ethical climate at EPA leaves something to be desired. The risk assessment of fluoride conducted in 1985, a case in point, far exceeds any previous unethical use of science at EPA. Failure to challenge this abuse would be shirking our duty as civil servants, defrauding the public (and harming the public health) and agreeing to become prostitutes for the aims of government politicians. To expose this abuse and establish mechanisms to insure that it does not happen again, NFFE Local 2050 is involved.

... The draft report of the National Toxicology Program (NTP) entitled the "Toxicology and Carcinogenesis Studies of Sodium Fluoride in F344/N Rats and B6C3F1 Mice" was released on April 2, 1990. On April 26, the report was "peer reviewed" at a public meeting in Research Triangle Park, North Carolina by the NTP Board of Scientific Counselors. The final report has not as yet been released.

The report concluded that there was "equivocal evidence of osteosarcoma" in male rats but not in female rats, or male or female mice. The term equivocal as it is used here means that the fluoride "may" cause cancer. This conclusion, which was concurred in with the peer review panel, represents a major change from the previous government position that fluoride is perfectly safe. This report was prepared by the staff of NTP. The study itself, however, was actually done by Battelle Columbus Laboratories under contract to NTP. A quality assurance contractor audited the Battelle report, and a separate group of pathologists reviewed the slides. Apparently the Board of Scientific Counselors did not receive the Battelle report to review nor the deliberations of the pathology work group. The board members, toxicologists, pathologists, and statisticians, reviewed only what they had in front of them in the technical report. Apparently they were not familiar with the fluoride literature...

... At the outset I failed to realize that the title of the draft report itself was misleading... the report actually contains the highly important results of a battery of genetic toxicology studies, as well as the studies of rats and mice but the title suggests that the report is concerned only with the health effects found during the 2 year animal studies. The findings of these genetic studies are minimized and obfuscated in the report. Although mentioned in the "Results" section on p. 79, their meaning is not elaborated upon in the "Discussion" section starting on p. 81. Finally, these studies are not mentioned in the "Conclusions" on p. 95, which is quite inexplainable because they show a positive relationship between fluroide exposure and genetic damage; they therefore lend support to the evidence for carcinogenicity. Dr. John Bucher, the study director himself, noted the significance of these genetic studies in a letter to the New York State Coalition Opposed to Fluoridation. His conclusions stated in the letter failed to appear in the NTP report.

Flaws in Cancer Study

The cancer study on rats and mice was conducted with a fair number of animals: 100 animals of each sex in the control and high dose groups and about 70 animals of each sex in the low and mid-dose groups. Ten animals of each group were sacrificed at 24 and 66 weeks, the remainder to be sacrificed at the end of two years. In other words there were actually 50 animals in the low and mid-dose groups, 80 in the high dose and control groups. Considering the importance of this study - the exposure of the entire US population to this substance - this was a minimal effort which becomes even more evident when the doses are considered. Both rats and mice received 11, 45, and 79 ppm fluoride in drinking water. Male rats received 0.55, 2.2, and 3.6 mg/kg/day (low doses compared to most cancer studies). When it was determined that the study could only be run with low doses, the number of animals should have been increased.

The most important observation: the control animals were not fed fluoride-free diets. Also, the animals were four weeks old at the beginning of the study and obviously not fluoride free. Male rats were fed a diet containing 7.9 ppm or 0.2 mg/kg/day of fluoride, about six times the dose received by a human drinking 2 liters/day of water containing 1 ppm fluoride. Animal studies have been successfully completed with a diet containing 0.05 ppm fluoride. The historical controls had even more fluoride in the diet. NTP estimates that normally their standard feed has from 28 to 47 ppm, or about 0.7 to 1.2 mg/g/day. The study had this to say about the dose concerning historical controls:

Assuming a maximum bioavailability of 60%, the historical database animals actually constitute a group receiving sufficient fluoride to place them between the low- and mid-concentration groups in the current 2-year studies.

This is an excellent argument for excludng the use of historical controls as a comparison group anywhere in the report. Yet, time and time again, they are used to compare with the results of the study and often it is to downgrade the results.

The study found osteosarcomas in the bone of three male rats in the high-dose group and in one male rat in the mid-dose group. These four tumors were found to occur with a statistically significant dose-response trend by the logistic regression test (p = 0.027). The three osteosarcomas in the high dose groups were also significantly higher than the rate of 0.6% of osteosarcomas and osteomas at all sites in control male rats in the historical database. Thus even though the historical controls were actually dosed with fluoride, the results at the high dose in this study were still significantly higher.

I should point out that another osteosarcoma was found in the subcutis of the flank of a male rat in the high dose group which was discounted because the radiographs showed no evidence of a primary neoplasm of the bone. Nevertheless one of the osteosarcomas which they did claim was not seen in the radiograph.

Still another soft tissue tumor described as "... collagenous connective tissue with well-defined islands of osteoid and/or woven bone" was ignored because they couldn't classify it.

Addng to the significance of this study is the level of fluoride found in the bone at the conclusion of the study. At the highest dose tested (79 ppm or 0.58 mg/kg/day as fluoride), the male rats had 5263 ppm of fluoride in bone ash. As noted in the report itself, this is within the range that has been found in humans exposed to less than the legal limit of 4 mg/L set by the EPA.

A number of other neoplasms were found to be elevated at various sites in the rats and mice. In the oral cavity, squamous cell papillomas and squamous cell carcinomas were found in the male rats as follows::: 0/80 in the controls, 1/51 at the low dose, 2/50 at the mid-dose and 3/80 in the high dose. These results, according to NTP, were marginal and not significantly greater than the controls. A margincal increase in thyroid neoplam in the high-dose group of male rats likewise was not considered significant. The incidence of malignant lymphomas in female mice was marginally increased relative to controls...2

Possible Downgrading of Results by NTP

The most disturbing aspect of this study: At the April 26 public hearing Dr. Yiamouyiannis and Dr. Mel Reuber pointed out that a number of cancerous and precancerous lesions, found by Battelle Columbus, were downgraded by a special Pathology Working Group hired by NTP to review the Battelle study. Dr. Reuber, a board certified pathologist and former employee of the National Cancer Institute, noted that hepatocholangiocarcinomas were downgraded to hepatoblastoma and finally to hepatocarcinoma. Upon reviewing the slides he stated that the pathology working group had erred.

Dr. Reuber's opinion is particularly significant because he was the one who first identified this lesion in the Journal of the National Cancer Institute. These rare liver tumors were found in mice, meaning that the study actually found cancer in both species which would have forced the NTP to change the equivocal rating of cancer to the next high category - "some evidence." Other tumors were also downgraded: adrenal pheochronocytomas to hyperplasias and liver adenomas to eosinophilic foci. Dr. Marcus and I have recommended to the EPA Administrator that EPA conduct an independent analysis of the Battelle study.

Summary

The possibility that fluoride causes cancer is much more likely than reported by NTP. The report itself seems to have been designed to downplay the results and therefore to mislead the public:

1. That all three genetic toxicology tests were positive adds support to the conclusion that fluroide may be carcinogenic.

2. The control groups actually received large amounts of fluroide in their feed, about 7.9 ppm, which caused the study to be less sensitive.

3. The historical controls, which were often used to downplay the results of this study, may have received 27 to 48 ppm in their feed. For this reason they should not have been used for comparison purposes.

4. There may have been a systematic downgrading of postive findings after Battelle Columbus reported the original data to NTP.

5. The public was excluded from any significant input into the review of this report.


From TOXNET

Cah Nutr Diet 1991;26(6):408-11

[Placental transfer of fluoride]

[In French]

Caldera R

Service de Pțediatrie B - Hìopital Saint Vincent de Paul, Paris, France

Gas chromatography was used to measure the maternal and fetal plasma inorganic fluoride values at term in 91 women. They were assigned to one of four groups: group A were untreated controls; group B received a single daily dose of 1.5 mg of fluoride (as calcium fluoride) during the final trimester of pregnancy; group C was given a single dose of 1.5 mg of fluoride (as sodium fluoride) and group D was given 2 daily doses of fluoride (as sodium fluoride) totaling 1.5 mg. There was a significant difference between the cord plasma fluoride levels of the newborns in the untreated group (mean: 27.8 ug/L) and that of the combined supplemented groups B, C and D (mean: 58.3 ug/L). There was no difference between the average fluoride levels in the three supplemented groups. In 17 women, gestation time between 19 and 34 weeks, fluoride levels were measured in the mother (mean: 17.11 ug/L) and in the foetus by cord tapping (mean: 35.64 ug/L). In 4 other mothers, was given a dose of 1 mg of sodium fluoride; one hour later, mean cord fluoride level was 86.5 ug/L. These results indicate that placental transfer of fluoride occurs early during pregnancy and that supplementation during the final trimester of pregnancy will significantly elevate cord plasma fluoride concentrations.


http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=2022784&dopt=Abstract

J Am Dent Assoc 1991 Apr;122(4):24

'Fluoride is safe.' Concludes DHHS study.

Publication Types: News

PMID: 2022784 [PubMed - indexed for MEDLINE]


Environmental Sciences 1 1991; 56-62

Chronic fluoride toxicity and pituitary-adrenal function

TK Das and AK Susheela

Reprints: TK Das, All India Institute of Medical Sciences, New Delhi, India

Some of the least-studied calcium-regulating hormones in chronic fluoride toxicity and fluorosis are the glucocorticoids. They are produced by the adrenal cortex and are known to play an important role in bone metabolism by regulating the calcium homeostasis. Hypocortisolemia in fluorosis patients and in experimental animals subjected to long-term fluoride administration has been reported. The present study was undertaken to determine the cause of adrenal insufficiency in chronic fluoride toxicity. Single-dose metyrapone and ACTH stimulation tests were applied. Both of the tests clearly demonstrated that in chronic fluoride toxicity, there is secondary adrenal insufficiency due to suppressed ACTH release system.


http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=1943439&dopt=Abstract

Life Sci 1991;49(17):1245-52

Interaction of aluminum ions with phosphoinositide metabolism in rat cerebral cortical membranes.

Candura SM, Castoldi AF, Manzo L, Costa LG.

Department of Environmental Health, University of Washington, Seattle 98195.

Aluminum (Al) is believed to exert a primary role in the neurotoxicity associated with dialysis encephalopathy and has been suggested to be involved in a number of other neurological disorders, including Alzheimer's disease. Al, complexed with fluoride to form fluoroaluminate (AlF4-), can activate the GTP-binding (G) proteins of the adenylate cyclase and retinal cyclic GMP phosphodiesterase systems. Since an involvement of G-proteins with cerebral phosphoinositide (PtdIns) metabolism has also been suggested, in this study we investigated the interaction of the stable GTP analogue GTP(S), Al salts and NaF with this system. In rat cerebral cortical membranes, GTP(S) dose-dependently stimulated [3H]inositol phosphates ([3H]InsPs) accumulation. This effect was potentiated by carbachol and was partially prevented by the GTP-binding antagonist GDP(S), indicating that CNS muscarinic receptor activation is coupled to PtdIns hydrolysis via putative G-protein(s). GTP(S) stimulation was also inhibited by phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C, which is known to exert a negative feedback control on agonist-stimulated PtdIns metabolism. Both Al salts and NaF mimicked the action of GTP(S) in stimulating PtdIns turnover. Their actions were highly synergistic, suggesting that AlF4- could be the active stimulatory species. However, the stimulatory effects of AlCl3 and/or NaF were not potentiated by carbachol and were not inhibited by GDP(S) and PMA, suggesting that separate sites of action might exist for GTP(S) and AlF4-. In the nervous tissue, activation of PtdIns hydrolysis by Al (probably as AlF4-) may be mediated by activating a regulatory G-protein at a location distinct from the GTP-binding site or by a direct stimulation of phospholipase C.

PMID: 1943439 [PubMed - indexed for MEDLINE]


http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=1664774&dopt=Abstract

Brain Res 1991 Nov 1;563(1-2):303-5

Soman reversibly decreases the duration of Ca2+ and Ba2+ action potentials in bullfrog sympathetic neurons.

Heppner TJ, Fiekers JF.

Department of Anatomy and Neurobiology, University of Vermont, College of Medicine, Burlington 05405.

Soman, (pinacoloxymethyl-phosphoryl fluoride) (0.1-10 microM) an irreversible cholinesterase inhibitor, reversibly reduced the duration of calcium (Ca2+)- and barium (Ba2+) spikes without significantly affecting spike amplitude in sympathetic postganglionic neurons of the adult bullfrog (Rana catesbeiana). The soman-induced shortening of the spike duration was not prevented by pretreatment with either (+)-tubocurarine (100 microM) or hexamethonium (100 microM) and atropine (10 microM) and was also recorded from acutely-dissociated sympathetic neurons. These results suggest that soman has a direct action to decrease calcium entry through voltage-dependent channels activated during a spike. This effect may contribute to both the decrease in the duration of the spike after-hyperpolarization (AHP) and the enhanced neuronal excitability produced by soman in these neurons.

PMID: 1664774 [PubMed - indexed for MEDLINE]


http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=1784417&dopt=Abstract

Neurosci Lett 1991 Nov 11;132(2):171-4

Interference of AlF4- with nucleotide and DNA binding of rat histone H1 in vitro. Implications for the pathogenesis of Alzheimer's disease.

Oikarinen J, Mannermaa RM, Tarkka T, Yli-Mayry N, Majamaa K.

Collagen Research Unit, University of Oulu, Finland.

We demonstrate here that the H2PO4- analogue AlF4- binds to the nucleotide-binding site of rat liver histone H1 in vitro, and interferes with nucleotide recognition and H1 DNA binding. AlF4- may thus compromise the genetically determined pattern of protein synthesis through binding to H1, the general repressor. The present findings are of interest as a number of studies have implicated aluminium as a factor in the pathogenesis of Alzheimer's disease.

PMID: 1784417 [PubMed - indexed for MEDLINE]


http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=1789596&dopt=Abstract

Ann N Y Acad Sci 1991;633:626-7

Fluoroacetate, a selective inhibitor of the glia tricarboxylic acid cycle, attenuated the release of luteinizing hormone-releasing hormone from the hypothalamus of ovariectomized rats.

Wu TJ, McArthur NH, Harms PG.

Department of Animal Science, Texas A&M University, College Station 77843.

PMID: 1789596 [PubMed - indexed for MEDLINE]


http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=1650708&dopt=Abstract

Exp Brain Res 1991;84(3):680-4

Brief exposure to the G-protein activator NaF/AlCl3 induces prolonged enhancement of synaptic transmission in area CAl of rat hippocampal slices.

Publicover SJ.

School of Biological Sciences, University of Birmingham, Edgbaston, UK.

Rat hippocampal slices were exposed briefly (12-15 min) to AlF4- (10 mmol/l NaF, 10 mumol/l AlCl3). The effect on synaptic transmission in area CAl was measured using extracellular electrodes placed in the stratum pyramidale and stratum radiatum. During fluoride exposure, both spike and EPSP amplitude fell to very low levels. Upon washout, spike amplitude recovered beyond control values, and in half of the preparations a prolonged enhancement of spike amplitude (greater than 2 h) occurred. Similar modulation of EPSP slope indicated that these charges were primarily synaptic. If Al3+ was omitted from the F(-)-containing saline, enhancement of spike amplitude, when observed, was brief (20-30 min) and no enhancement of EPSP slope was seen. Omission of Ca2+ from the AlF(4-)-containing saline also abolished any long-lasting enhancement of synaptic transmission, though population spike amplitude in most slices showed a brief (20-30 min) stimulatory response. In preparations in which LTP had previously been saturated, synaptic transmission was not enhanced by exposure to AlF4-. It is concluded that NaF/ACl3 exposure induces an LTP-like process by G-protein activation, which involves recruitment of processes involved in LTP, possibly including an enhancement of Ca(2+)-influx.

PMID: 1650708 [PubMed - indexed for MEDLINE]


http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=1756866&dopt=Abstract

FEBS Lett 1991 Dec 9;294(3):239-43

Trimeric G-proteins of the trans-Golgi network are involved in the formation of constitutive secretory vesicles and immature secretory granules.

Barr FA, Leyte A, Mollner S, Pfeuffer T, Tooze SA, Huttner WB.

Cell Biology Programme, European Molecular Biology Laboratory, Heidelberg, Germany.

Non-hydrolysable analogues of GTP, such as GTP gamma S and GMP-PNP, have previously been shown to inhibit the formation of constitutive secretory vesicles (CSVs) and immature secretory granules (ISGs) from the trans-Golgi network (TGN). Using a cell-free system, we show here that the formation of these vesicles is also inhibited by [A1F4]-, a compound known to act on trimeric G-proteins. Addition of highly purified G-protein beta gamma subunits stimulated, in a differential manner, the cell-free formation of both CSVs and ISGs. ADP-ribosylation experiments revealed the presence of a pertussis toxin-sensitive G-protein alpha subunit in the TGN. We conclude that trimeric G-proteins regulate the formation of secretory vesicles from the TGN.

PMID: 1756866 [PubMed - indexed for MEDLINE]


http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=1957170&dopt=Abstract

Science 1991 Nov 22;254(5035):1197-9

Binding of ARF and beta-COP to Golgi membranes: possible regulation by a trimeric G protein.

Donaldson JG, Kahn RA, Lippincott-Schwartz J, Klausner RD.

Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892.

The binding of cytosolic coat proteins to organelles may regulate membrane structure and traffic. Evidence is presented that a small guanosine triphosphate (GTP)-binding protein, the adenosine diphosphate ribosylation factor (ARF), reversibly associates with the Golgi apparatus in an energy, GTP, and fungal metabolite brefeldin A (BFA)-sensitive manner similar to, but distinguishable from, the 110-kilodalton cytosolic coat protein beta-COP. Addition of beta gamma subunits of G proteins inhibited the association of both ARF and beta-COP with Golgi membranes that occurred upon incubation with guanosine 5'-O-(3-thiotriphosphate) (GTP-gamma-S). Thus, heterotrimeric G proteins may function to regulate the assembly of coat proteins onto the Golgi membrane.

PMID: 1957170 [PubMed - indexed for MEDLINE]


http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=1648693&dopt=Abstract

Neurosci Lett 1991 Apr 1;124(2):242-5

Melatonin receptor mRNA expression in Xenopus oocytes: inhibition of G-protein-activated response.

Fraser SP, Barrett P, Djamgoz MB, Morgan PJ.

Department of Biology, Imperial College of Science, Technology and Medicine, London, U.K.

Melatonin is the major endocrine product of the pineal gland in the mammalian brain and plays a variety of roles in photoperiodic functions. In order to investigate melatonin receptors, poly(A)+ RNA was extracted from pars tuberalis of the ovine pituitary and injected into oocytes of Xenopus laevis. After 3-5 days of incubation, functional melatonin receptors were expressed. Receptors were revealed by their inhibitory effect upon oscillatory currents resulting from AlF4-induced activation of G-proteins in the oocyte membrane under voltage clamp conditions. The effect of melatonin was dose-dependent, non-desensitizing and was not observed in uninjected oocytes.

PMID: 1648693 [PubMed - indexed for MEDLINE]


http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=1657021&dopt=Abstract

J Mol Endocrinol 1991 Oct;7(2):137-44

Intracellular signalling in the ovine pars tuberalis: an investigation using aluminium fluoride and melatonin.

Morgan PJ, Hastings MH, Thompson M, Barrett P, Lawson W, Davidson G. Rowett

Research Institute, Bucksburn, Aberdeen.

The effect of aluminium fluoride (AlF4-) has been studied on inositol phosphate accumulation, calcium mobilization, cyclic AMP production and [2-125I]iodomelatonin binding in ovine pars tuberalis cells. These cells have high-affinity receptors for, and respond to, melatonin through inhibition of forskolin-stimulated adenylate cyclase. In the presence of 10 mM LiCl, AlF4- stimulated the net accumulation of inositol monophosphate and inositol bisphosphate. Consistent with these findings, AlF4- increased intracellular calcium; although this response was attenuated in calcium-depleted medium, indicating that the calcium response comprises both intracellular and extracellular components. Melatonin was ineffective on either basal or AlF4(-)-stimulated turnover of inositol phosphates. In concordance with the inositol phosphate response, melatonin had no effect on either the AlF4(-)-stimulated or the basal calcium levels. AlF4- blocked the increase in cyclic AMP stimulation by 1 microM forskolin, being as effective as melatonin, achieving approximately 90% inhibition. AlF4- also attenuated the binding of [2-125I]iodomelatonin to ovine pars tuberalis membranes by 15%. At the concentration used, these results are consistent with the interpretation that AlF4- activates many G protein-mediated responses, and thus imply that the inhibitory pathway for cyclic AMP predominates over the stimulatory arm, whereas there can only be a stimulatory pathway linked to phosphoinositide metabolism in ovine pars tuberalis cells.

PMID: 1657021 [PubMed - indexed for MEDLINE]


http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=1661824&dopt=Abstract

Brain Res Mol Brain Res 1991 Oct;11(3-4):265-71

Altered phosphoinositide-specific phospholipase C and adenylyl cyclase in brain cortical membranes of cats with GM1 and GM2 gangliosidosis.

Claro E, Wallace MA, Fain JN, Nair BG, Patel TB, Shanker G, Baker HJ.

Department of Biochemistry, University of Tennessee, Memphis 38163.

Phosphoinositide-specific phospholipase C and adenylyl cyclase were studied in brain cortical membranes from cats with GM1 and GM2 gangliosidosis. In contrast to brain cortical membranes from unaffected control cats, phospholipase C acting against exogenously supplied phosphoinositide substrates did not respond to stimulation by GTP gamma S, carbachol or fluoroaluminate in cortical membranes of cats with gangliosidosis. However, the enzyme was activated by calcium in membranes from affected cats to the same extent as in membranes from control cats. Basal adenylyl cyclase activity was increased 3-fold in cortical membranes of cats with GM1 and GM2 gangliosidosis, compared with unaffected sibling controls. Fluoroaluminate was equally effective in stimulating adenylyl cyclase in controls and in membranes of affected and normal cats. In addition, GppNHp was able to inhibit the forskolin-activated enzyme both in membranes from cats with gangliosidosis and sibling controls. These data suggest that the activation of phosphoinositide-specific phospholipase C in brain membranes by guanine nucleotide binding proteins is markedly impaired in GM1 and GM2 gangliosidoses.

PMID: 1661824 [PubMed - indexed for MEDLINE]


http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=1773667&dopt=Abstract

Zhonghua Yu Fang Yi Xue Za Zhi 1991 Sep;25(5):269-71

[The clinical features of 160 cases of acute sodium silicon fluoride poisoning]

[Article in Chinese]

Dong YT.

First People's Hospital of Wenling County, Zhejiang.

160 cases of acute sodium silicon fluoride poisoning due to accidental intake are reported in this paper. Owing to the great amount of the chemical taken, the onset was abrupt and the digestive symptoms were prominent and severe. Twelve cases had acute gastritis or acute ulceration as shown by gastroscopy. The cardiovascular findings were notable. Ninety-six cases had abnormal electro-cardiographic findings. Abnormal heart rhythm and S-T segment changes were the main findings. These changes were positively correlated with the toxin amount of fluoride. Two cases died from sudden arrest of heart beat. Lung, kidney, liver, and brain functions were also impaired. The authors suggested diagnostic criteria and control procedures.

PMID: 1773667 [PubMed - indexed for MEDLINE]


http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=1796315&dopt=Abstract

Sheng Li Xue Bao 1991 Oct;43(5):512-7

[An experimental study of inhibition on lactation in fluorosis rats]

[Article in Chinese]

Yuan SD, Song KQ, Xie QW, Lu FY.

Neuroendocrine Research Laboratory, China Medical University, Shenyang.

The effect of fluorosis on lactation, lactotroph function and ultrastructure were studied in lactating rats. The results were as follows:
1) Inhibition of lactation in lactating rats with chronic fluorosis was assessed by stunting growth of pups and decrease in the amount of milk suckled by pups in 30 min. Metoclopramide, a blocker of dopamine receptor, could improve lactation in these rats.
2) During chronic fluorosis serum PRL level was decreased, however, PRL content in pituitary was increased. Electronmicroscopic examination showed accumulation of large mature secretory granules and appearance of extremely large abnormal secretory granules in lactotroph cytoplasma. These findings indicate that hormone release of pituitary lactotrophs is obstructed in lactating rats with fluorosis, and the toxic effect of fluoride is mediated by an enhanced function of dopaminergic system in hypothalamus.

PMID: 1796315 [PubMed - indexed for MEDLINE]


http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=1886094&dopt=Abstract

J Reprod Fertil 1991 Jul;92(2):353-60

A study of the effect of high concentrations of fluoride on the reproductive organs of male rabbits, using light and scanning electron microscopy.

Susheela AK, Kumar A.

Department of Anatomy, All India Institute of Medical Sciences, New Delhi.

Fluoride was orally administered to rabbits at 10 mg NaF/kg body weight for 18 or 29 months. The animals were then killed and the structure of the testis, epididymis and vas deferens studied under light and scanning electron microscopes. In animals treated for 29 months, the spermatogenic cells in the seminiferous tubules were disrupted, degenerated and devoid of spermatozoa. In animals treated for 18 or 29 months, loss of cilia on the epithelial cells lining the lumen of the ductuli efferentes of the caput epididymidis and of stereocilia on the epithelial cells lining the lumen of the vas deferens was observed. In some regions of the epithelial lining of the lumen of the ductuli efferentes and vas deferens, the boundaries of the cells were not clear and appeared to be peeled off. Mucus droplets were abundant in the vas deferens of control animals, but absent in both the treated groups. Spermatogenesis ceased only in animals treated for 29 months. The difference in the structural changes observed in the testes of the 2 treated groups may have been due to the blood-testis barrier. It is concluded that ingestion of high concentrations of fluoride has harmful effects on the male reproductive system.

PMID: 1886094 [PubMed - indexed for MEDLINE]


http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=1803346&dopt=Abstract

Pharmacol Toxicol 1991 Nov;69(5):330-7

Aluminofluoride- and epidermal growth factor-stimulated DNA synthesis in MOB 3-4-F2 cells.

Kawase T, Orikasa M, Suzuki A.

Department of Pharmacology, Niigata University School of Dentistry, Japan.

In attempt to study the mechanism of F(-)-induced, osteoblast-mediated bone formation, we tried to show the characteristics of Al-F complex-induced mitogenesis in osteoblastic cells. The MOB 3-4-F2 cell line, an osteoblast-like cell line derived from neonatal mouse calvaria, responded to F- (1-2 mM) combined with Al3+ and epidermal growth factor (EGF, 0.01-100 ng/ml) with increased DNA synthesis. Of the several types of Al-F complexes, AlF4- is thought to act as a mitogenic factor. On the other hand, NaF at high concentrations (greater than 2 mM) markedly decreased cell viability. The AlF(4-)-stimulated DNA synthesis at least with a delay of 48 hr, while EGF stimulated DNA synthesis within a few hours (4-6 hr). Both 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7) and staurosporine, inhibitors of protein kinase C (PKC), further enhanced DNA synthesis in AlF(4-)-treated cells, whereas 12-O-tetradecanoyl-13-acetate (TPA), an activator of PKC, decreased the DNA synthesis. In EGF-treated cells, staurosporine and TPA, but not H-7, decreased DNA synthesis. In addition, indomethacin, an inhibitor of cyclooxygenase, partly inhibited the EGF-induced mitogenesis, which, however, was restored by addition of PGE2. AlF4-, as well as EGF, stimulated the release of arachidonic acid and its metabolites. Indomethacin failed to inhibit the AlF(4-)-induced mitogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)

PMID: 1803346 [PubMed - indexed for MEDLINE]


http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=1839778&dopt=Abstract

Reprod Toxicol 1991;5(6):505-12

Microdose vasal injection of sodium fluoride in the rat.

Chinoy NJ, Rao MV, Narayana MV, Neelakanta E.

Department of Zoology, University School of Sciences, Gujarat University, Ahamadabad, India.

A single microdose (50 micrograms/50 microL) injection of sodium fluoride (NaF) into the vasa deferentia of adult male albino rats (Rattus norvegicus) caused arrest of spermatogenesis and absence of spermatozoa in the lumina of the seminiferous tubules of the testes, which consequently led to a decline in the sperm count in the caudae epididymides. Scanning electron microscopy of cauda and vas deferens sperm revealed deflagellation and tail abnormalities. This is probably related to the alterations in the internal milieu of these organs which rendered the spermatozoa immotile and consequently caused fertility impairment in the experimental animals. Thus microdoses of sodium fluoride were found to affect reproductive function and fertility rate.

PMID: 1839778 [PubMed - indexed for MEDLINE]


http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=1747106&dopt=Abstract
Biochem J 1991 Dec 1;280 ( Pt 2):335-40

Fluoroaluminate treatment of rat liver microsomes inhibits GTP-dependent vesicle fusion.

Comerford JG, Dawson AP.

School of Biological Sciences, University of East Anglia, Norwich, U.K.

1. Inhibition of GTP-dependent membrane fusion of rat liver microsomes requires preincubation of the membranes with GDP (17 microM) and relatively high Mg2+ concentration (0.5 mM) as well as AlCl3 (30 microM) and KF (5 mM). Preincubation is required for maximal inhibition (75%).
2. Vesicle fusion in rat liver microsomes has been demonstrated in the absence of polyethylene glycol (PEG). Further, inhibition by AlF4- of GTP-dependent vesicle fusion in the absence of PEG has been demonstrated.
3. Under similar preincubation conditions AlF4- can bring about inhibition (80%) of the high-affinity PEG-stimulated GTPase activity in rat liver microsomes, previously described by Nicchitta, Joseph & Williamson [(1986) FEBS Lett. 209, 243-248].
4. Preincubation of small-Mr GTP-binding proteins (Gn proteins) on nitrocellulose strips with GDP (20 pM), AlCl3 (30 microM) and KF (5 mM) results in inhibition of binding of guanosine 5'-[gamma-[35S]thio]triphosphate to Gn proteins. The extent of inhibition of this binding differs for different Gn proteins.

PMID: 1747106 [PubMed - indexed for MEDLINE]


Fluoride 1991; 24(1):29-39

Effects of vitamin C and calcium on the reversibility of fluoride-induced alterations in spermatozoa of rabbits

Chinoy NJ *, Sequeira E, Narayana MV

* Department of Zoology, University School of Sciences, Gujarat University, Ahmedabad, India

Summary: The present study was designed to investigate the effects of fluoride on the metabolism and functions of cauda epididymal spermatozoa of rabbits. The studies on reversibility of fluoride-induced effects by fluoride withdrawal, Vitamin C (ascorbic acid, AA) or Calcium (Ca+2) and combined AA + CA+2 ingestion were also investigated.

Adult Male rabbits, Group II and III, were fed 20 and 40 mg/kg body weight sodium fluoride, respectively, for 30 days. Afterwards, cauda epididymal spermatozoa were obtained by micropuncture technique. Alterations in the activities of some specific androgen-dependent enzymes on sperm namely, ATPase, ACP, SDH, and protein, as well as reduction in Na+ and K+ levels in the spermatozoa, might be due to altered and hostile internal milieu of the epididymis in NaF-treated rabbits. Reduction in sperm motility, count, and changes in their morphology and metabolism led to the significant decline in fertility of the treated animals. After withdrawal of NaF treatment for 30 days (Group IV), no recovery was obtained in all the parameters which were altered, including fertility rates.

During the withdrawal period, AA or CA+2 and combined AA + Ca+2 were administered to Groups V, VI, and VII, respectively. With both AA or CA+2 marked recovery ocurred from all induced effects. The extent of recovery, however, was somewhat more pronounced by AA treatment than that brought about by CA+2 administration during the withdrawal period. The combined treatment with AA + CA+2 manifested a synergistic effect for recovery of all parameters. The effects of fluoride are therefore transient and reversible, in agreement with earlier data. Moreover, AA and Ca+2 have therapeutic importance in fluorotic animals. These findings have a direct bearing on human subjects exposed to high fluroide levels.


Fluoride 1991; 24(1):23-28

Effect of chronic fluoride toxicity on glucocorticoid levels in plasma and urine

TK Das and AK Susheela *

* Dr. AK Susheela, Fluoride and Fluorosis Research Laboratories, Department of Anatomy, All India Institute of Medical Sciences, New Delhi, India

Summary: Circulating levels of cortisol were estimated in 14 fluorosis patients and 9 healthy control subjects. The plasma cortisol level was markedly dereased in the patient group. To confirm this, the protocol was repeated in fluroide-treated experimental animals with sex and age matched controls. The fluoride-treated group also demonstrated marked hypocortisolemia, and the decrease was consistent at different time periods irrespective of diurnal variation. The 17-hydroxycorticosteroid levels in urine was also decreased (p < .001), clearly suggesting adrenal hypofunction in chronic fluoride toxicity.

Excerpt: Glucocorticoids are one of the least studied calcium regulating hormones in chronic fluoride toxicity and fluorosis. At supraphysiologic level, they are known to inhibit bone formation and stimulate bone resporption, resulting in severe bone loss or osteopenia (1-3). Glucocorticoid are also known to inhibit intestinal calcium absorption (4), possibly by inhibiting the synthesis of a calcium binding protein in intestine (5). Enhanced urinary calcium excretion is also very common in subjects having higher circulating levels of endogenously producd corticosteroid (6), or in patients under prolonged glucocorticoid therapy (7)...

1) Bressot C et al (1979). Histomorphometric profile, pathophysiology and reversibility of corticosteroid induced osteoporosis. Metab Bone Dis Rel Res 1:303.
2) Raiz LG et al ( (1972). Effect of glucocorticoids on bone resorption in tissue culture. Endocrinology 90:961-967.
3) Peck W et al (1984). Corticosteroid and Bone. Calcif Tissue Int 36:4-7.
4) Klein RG et al (1977). Calcium absorption in exogenous hypercortisonism. J Clin Invest 60:253-259.
5) Feher JJ et al (1979). Intestinal calcium binding protein and calcium absorption in cortisol treated chicks: vitamins and 1,25 dihydroxy vitamim D3. Endocrinology 104:547-551.
6) Jowsey J et al (1970). Bone formation in hypercortisonism. Acta Endocrin 63:21-28.
7) Pechet MM et al (1959). Metabolic studies with a new series of 1,4-dienesteroids. II. Effects in normal subjects of prehnisone, prednisolone and 9a-fluoroprednisolone. J Clin Invest 38:691-701.


http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=1744080&dopt=Abstract

J Biol Chem 1991 Dec 5;266(34):22872-7

Mechanism of fluoride activation of G protein-gated muscarinic atrial K+ channels.

Yatani A, Brown AM.

Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030.

Aluminum fluoride (AlF4-) activates the heterotrimeric G protein Gs (stimulatory G protein of adenylylcyclase) (Sternweis, P. C., and Gilman, A. G. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 4888-4891) and GT (transducin), and for GT, Bigay et al. (Bigay, J., Deterre, P., Pfister, C., and Chabre, M. (1985) FEBS Lett. 191, 181-185) have made the intriguing proposal that AlF4- acts by mimicking the gamma-phosphate of GTP. The endogenous G protein (probably G alpha i-2 or G alpha i-3 (Yatani, A., Mattera, R., Codina, J., Graf, R., Okabe, K., Padrell, E., Iyengar, R., Brown, A. M., and Birnbaumer, L. (1988) Nature 336, 680-682) that stimulates the muscarinic atrial K+ (K+[ACh]) channel is also thought to be activated by AlF4- (Kurachi, Y., Nakajima, T., and Ito, H. (1987) Circulation 76, 105P). To investigate the AlF4- mechanism, we applied potassium fluoride (KF) to the cytoplasmic face of inside-out membrane patches excised from guinea pig atria. We found that KF activated single K+[ACh] channel currents in both a concentration- and a Mg(2+)-dependent manner. Activation persisted following removal of KF, but unlike activation by guanosine 5'-(3-thiotriphosphate) (GTP gamma S), was fully reversed by removal of Mg2+. Evidence for Al3+ involvement was that the Al3+ chelator deferoxamine (500 microM) inhibited KF activation and that at low concentrations of KF (less than 1 mM), micromolar AlCl3 concentrations potentiated KF stimulation. The rate of activation produced by KF was far slower than the rate produced by GTP or GTP gamma S, and unlike these guanine nucleotides, the rate was unchanged in the presence of agonist. To test the gamma-phosphate-mimicking hypothesis, we evaluated the requirement for GDP; and to accomplish this, it was necessary to establish a condition that ensured exchange of guanine nucleotides. This condition was satisfied by using the muscarinic agonist carbachol because both the rate and the extent of activation of the K+[ACh] channels produced by GTP were much faster in carbachol, and both were greatly slowed when GDP was added along with GTP. By contrast, the effects of KF were unchanged by carbachol in the presence or absence of GDP. Further evidence that GDP is not essential for activation by AlF4- was provided by the observation that during carbachol activation and following extensive washing with GMP, guanosine 5'-O-(2-thiodiphosphate) at blocking concentrations had no effect on activation produced by KF.(ABSTRACT TRUNCATED AT 400 WORDS)

PMID: 1744080 [PubMed - indexed for MEDLINE]


http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=2043770&dopt=Abstract

Blood 1991 Jun 15;77(12):2746-56

Diacylglycerol generation in fluoride-treated neutrophils: involvement of phospholipase D.

English D, Taylor G, Garcia JG.

Department of Medicine, Indiana University School of Medicine, Indianapolis.

Neutrophils exposed to fluoride ion (F-) respond with a delayed and sustained burst of superoxide anion release that is both preceded by and dependent on the influx of Ca2+ from the extracellular medium. The results of this study demonstrate a similarly delayed and sustained generation of 1,2-diglyceride in F(-)-treated neutrophils, over 90% of which was 1,2-diacylglycerol. Diacylglycerol generation was not dependent on the presence of extracellular Ca2+. Conversely, in contrast to results obtained with other agonists, removal of extracellular Ca2+ markedly potentiated synthesis of diacylglycerol in F(-)-treated neutrophils. This effect was accompanied by a corresponding decrease in the recovery of phosphatidic acid. In either the presence or absence of extracellular Ca2+, phosphatidic acid accumulated before diacylglycerol in F(-)-treated cells, suggesting the latter was derived from the former. Consistent with this hypothesis, the phosphatidic acid phosphohydrolase inhibitor, propranolol, suppressed generation of diacylglycerol as it potentiated the accumulation of phosphatidic acid in F(-)-treated neutrophils. This effect was observed both in the presence and absence of extracellular Ca2+. Moreover, high levels of propranolol (160 mumol/L) effected complete inhibition of diacylglycerol generation in F(-)-treated neutrophils with a corresponding increase in phosphatidic acid generation. Phosphatidylethanol accumulated in neutrophils stimulated with F- in the presence of ethanol. The extent of phosphatidylethanol accumulation at all time points after addition of F- corresponded to decreased levels of both phosphatidic acid and diacylglycerol, indicating that phosphatidylethanol was derived from the phospholipase D-catalysed transphosphatidylation reaction. The results indicate that F- activates a Ca(2+)-independent phospholipase D, which appears to be the major, if not sole, catalyst for both phosphatidic acid and diacylglycerol generation in F(-)-treated neutrophils. Ca2+, mobilized as a result of F- stimulation and possibly as a consequence of phospholipase D activation, exerts a profound effect on cellular second messenger levels by modulating the conversion of phosphatidic acid to diacylglycerol.

PMID: 2043770 [PubMed - indexed for MEDLINE]


http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=1654060&dopt=Abstract

Am J Respir Cell Mol Biol 1991 Aug;5(2):113-24

Sodium fluoride induces phosphoinositide hydrolysis, Ca2+ mobilization, and prostacyclin synthesis in cultured human endothelium: further evidence for regulation by a pertussis toxin-insensitive guanine nucleotide-binding protein.

Garcia JG, Dominguez J, English D.

Department of Medicine, Indiana University School of Medicine, Indianapolis 46202.

The role of guanine nucleotide-binding proteins in the induction of prostacyclin synthesis by stimulated endothelial cells is incompletely understood. We report that sodium fluoride (NaF), a potent activator of cellular guanine nucleotide-binding proteins, affected time- and concentration-dependent generation of prostacyclin (PGI2) by cultured human umbilical vein endothelial cells without evidence of cellular toxicity detected by 51Cr or lactate dehydrogenase release. PGI2 synthesis by NaF-stimulated endothelial cells was associated with increases in arachidonate release, phosphoinositide hydrolysis, generation of inositol phosphates, and accumulation of diacylglycerol. These responses to NaF, as well as alpha-thrombin-mediated responses, were not dependent upon the availability of extracellular free Ca2+ but were associated with the mobilization of stored intracellular Ca2+ detected by the luminescence of the photoprotein aequorin. Neither PGI2 synthesis nor Ca2+ responses following alpha-thrombin or NaF stimulation were inhibited by pretreatment of cells with the islet activating protein from Bordetella pertussis but were significantly attenuated by the G protein inhibitor GDP beta S in permeabilized cells. Our results are compatible with a model wherein NaF directly activates a phosphoinositidase-linked guanine nucleotide regulatory protein, Gp, in human umbilical vein endothelial cell monolayers. This activation results in phosphoinositide hydrolysis, Ca2+ mobilization, arachidonate release, and subsequent functional activation, assessed by PGI2 release. Biologically relevant agonists such as alpha-thrombin may exert their influence on arachidonate metabolism, in part, by promoting receptor-dependent activation of this G protein.

PMID: 1654060 [PubMed - indexed for MEDLINE]


http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=1652206&dopt=Abstract

Am J Physiol 1991 Aug;261(2 Pt 2):F318-27

Fluoride mobilizes intracellular calcium and promotes Ca2+ influx in rat proximal tubules.

Dominguez JH, Garcia JG, Rothrock JK, English D, Mann C.

Nephrology Section, Veterans Administration Medical Center, Indianapolis, Indiana 46202.

In the renal proximal tubule, external Ca2+ ([Ca2+]o) is required for parathyroid hormone to elevate cytosolic Ca2+ ([Ca2+]i). However, other hormones increase [Ca2+]i in the absence of [Ca2+]o. These differences may arise from a diversity of signal transduction pathways acting on external and internal Ca2+ pools. However, Ca2+ influx may be necessary to expedite and maintain the rise of [Ca2+]i for a period after the initial surge. In this study, F- was used to probe the roles of intracellular Ca2+ mobilization, Ca2+ influx, and phosphoinositide (PI) hydrolysis on the surge of [Ca2+]i in rat proximal tubules. In the presence of external Ca2+; 1-20 mM F- evoked incremental rises of [Ca2+]i in tubules loaded with aequorin. Whereas 10 mM F- increased [Ca2+]i in the absence of [Ca2+]o, the time constant for the [Ca2+]i surge was increased. These findings are consistent with a role of Ca2+ influx on the effect of F- on [Ca2+]i. Indeed, 10 mM F- also enhanced the uptake of 45Ca2+, and promoted Ca2+ influx in aequorin- and fura-2-loaded, Ca(2+)-deprived tubules. In tubules, F- also activated PI hydrolysis with a time course that paralleled Ca2+ mobilization. The effect of F- on [Ca2+]i was not altered when the 39-kDa pertussis toxin substrate was inactivated with the toxin. This G protein was most likely Gi, because prostaglandin E2, an activator of Gi in tubules, dissociated the pertussis toxin-sensitive protein. The results support the notion that activation of a signal-transduction complex, the F- substrate, causes Ca2+ influx, mobilizes internal Ca2+, and activates PI hydrolysis in rat proximal tubules.(ABSTRACT TRUNCATED AT 250 WORDS)

PMID: 1652206 [PubMed - indexed for MEDLINE]


http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=1798747&dopt=Abstract

Photochem Photobiol 1991 Nov;54(5):703-7

The effect of fluoride on binding and photodynamic action of phthalocyanines with proteins.

Ben-Hur E, Dubbelman TM, Van Steveninck J.


Sylvius Laboratory, Department of Medical Biochemistry, Leiden, The Netherlands.

Fluoride inhibits chloroaluminum phthalocyanine tetrasulfonate (AlPcS)-induced photohemolysis when added to dye loaded cells prior to light exposure. The mechanism by which F- exerts this effect was studied by measuring the binding of phthalocyanine (Pc) to various proteins in the absence and presence of F-. Parallel measurements were made of the photodynamic action under these conditions. Fluoride reduced the binding to proteins of AlPcS and CoPcS. The binding of CuPcS, ZnPcS and H2PcS was not affected. When bound to bovine serum albumin and exposed to light, H2Pc, ZnPc and AlPcCl were bleached at a biphasic rate. Only the photobleaching of AlPcCl was affected by F-. The effect of F- was to inhibit the initial rapid phase without affecting the slower phase. In the presence of D2O only the second phase of photobleaching was enhanced, in the absence or presence of F-. No effect of F- was observed on tryptophan photooxidation or glyceraldehyde-3-phosphate dehydrogenase photoinactivation by AlPcS. Crosslinking of spectrin monomers photosensitized by AlPcS was inhibited by F- in parallel with the reduced binding of dye to the protein. It is concluded that F- exerts its effect by complexing with metal ligands of Pc. As a result, the dye may be released from the protein or the binding mode may be changed in such a way that effective photochemistry is prevented. Primary photophysical processes of Pc most probably are not affected by F-.

PMID: 1798747 [PubMed - indexed for MEDLINE]


http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=1800124&dopt=Abstract

Eur J Pharmacol 1991 Oct 15;203(2):311-4

Stimulation of miniature end-plate potential frequency by fluoride may involve activation of protein kinase C.

Light PE, Publicover SJ.

School of Biological Sciences, University of Birmingham, Edgbaston, U.K.

NaF caused a dose-dependent rise in miniature end-plate potential (MEPP) frequency at the frog neuromuscular junction. The effects on MEPP frequency of both NaF and the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) were rapidly reversed by the protein kinase C (PKC) inhibitor polymyxin B (2 microM). Theophylline augmented the response of MEPP frequency to TPA. It is concluded that the effect of fluoride on MEPP frequency may be through activation of phospholipase C and consequent PKC stimulation, and that the synergistic interaction of NaF and theophylline is consistent with such a mode of action.

PMID: 1800124 [PubMed - indexed for MEDLINE]


http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=1836355&dopt=Abstract

Biochim Biophys Acta 1991 Nov 18;1070(1):83-91

Inhibitory and stimulatory effects of fluoride on the calcium pump of cardiac sarcoplasmic reticulum.

Narayanan N, Su N, Bedard P.

Department of Physiology, University of Western Ontario, London, Canada.

While studying the effects of membrane phosphorylation on active Ca2+ transport in cardiac sarcoplasmic reticulum (SR) we used NaF (a conventional phosphatase inhibitor) in the Ca2+ transport assay medium to suppress protein dephosphorylation by endogenous phosphatases. Unexpectedly, depending on the experimental conditions employed, NaF was found to cause a strong inhibitory or stimulatory effect on ATP-dependent, oxalate-facilitated Ca2+ uptake (Ca2+ pump) activity of SR. Investigation of this phenomenon using canine cardiac SR revealed the following. Exposure of SR to NaF in the absence of Ca2+ or ATP in the Ca2+ transport assay medium (prior to initiating Ca2+ transport by the addition of Ca2+ or ATP) promoted a striking concentration-dependent inhibitory effect of NaF (50% and 90% inhibition with approx. 4 and 10 mM NaF, respectively) on Ca2+ uptake by SR; the magnitude of inhibition did not differ appreciably with varying oxalate concentrations. In contrast, exposure of SR to NaF in the presence of both Ca2+ and ATP resulted in a concentration-dependent stimulatory effect of NaF (half-maximal stimulation at approx. 2.5 mM NaF with 2.5 mM oxalate in assay) on Ca2+ uptake; the magnitude of stimulation decreased with increasing oxalate concentration (greater than 2-fold at 1 mM oxalate, 10% at 5 mM oxalate). The inhibitory effect prevailed when SR was exposed to NaF in the presence of Ca2+ alone (without ATP) or ATP alone (without Ca2+). Both the inhibitory and stimulatory effects of NaF were specific to fluoride ion, as NaCl (1-10 mM) showed no effect on Ca2+ uptake by SR under identical assay conditions. A persistently less active state of the Ca2+ pump (evidenced by decreased Ca2+ transport rates) resulted upon pretreatment of SR with NaF in the absence of Ca2+ or ATP; presence of Ca2+ and ATP during pretreatment prevented this transition. The inhibitory action of NaF on the Ca2+ pump was accompanied by a two-fold increase in K0.5 for Ca2+ and decrements in Hill coefficient (nH) and Ca(2+)-stimulated ATP hydrolysis, as well as steady-state level of Ca(2+)-induced phosphoenzyme. The stimulatory effect of NaF, on the other hand, was associated with an increase in the ratio of Ca2+ transported/ATP hydrolysed with only minor changes, if any, in the above parameters. These findings imply that the divergent effects of fluoride are dependent on specific conformational states of the Ca(2+)-ATPase which evolve during the catalytic and ion transport cycle.(ABSTRACT TRUNCATED AT 400 WORDS)

PMID: 1836355 [PubMed - indexed for MEDLINE]


http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=1660001&dopt=Abstract

FEBS Lett 1991 Nov 18;293(1-2):169-72

A novel conformationally restricted protein kinase C inhibitor, Ro 31-8425, inhibits human neutrophil superoxide generation by soluble, particulate and post-receptor stimuli.

Muid RE, Dale MM, Davis PD, Elliott LH, Hill CH, Kumar H, Lawton G, Twomey BM, Wadsworth J, Wilkinson SE, et al.

Department of Pharmacology, University College London, England.

A novel, bis-indolylmaleimide, Ro 31-8425, bearing a conformationally restricted side chain, inhibits protein kinase C isolated from rat brain and human neutrophils with a high degree of selectivity over cAMP-dependent kinase and Ca2+/calmodulin-dependent kinase. It also inhibits phorbol ester-induced intracellular events known to be mediated by protein kinase C (p47 phosphorylation in intact platelets, CD3 and CD4 down-regulation in T-cells). Ro 31-8425 inhibited superoxide generation in human neutrophils activated by both receptor stimuli (formyl-methionyl-leucylphenylalanine, opsonized zymosan, IgG and heat aggregated IgG) and post-receptor stimuli (1,2-dioctanoylglycerol and fluoride). The compound also blocked antigen driven, but not IL-2 induced, T-cell proliferation. These results support a central role for protein kinase C in the activation of the respiratory burst and antigen-driven T-cell proliferation.

PMID: 1660001 [PubMed - indexed for MEDLINE]


http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=1899863&dopt=Abstract

NOTE: AG Gilman, co-author of this report, shared the 1994 Nobel Prize for the discovery of "G-proteins and the role of these proteins in signal transduction in cells."

J Biol Chem 1991 Feb 25;266(6):3396-401

19F and 31P NMR spectroscopy of G protein alpha subunits. Mechanism of activation by Al3+ and F-.

Higashijima T, Graziano MP, Suga H, Kainosho M, Gilman AG.

Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235.

19F and 31P NMR spectroscopy was used to study the mechanism of activation of the alpha subunits of guanine nucleotide-binding regulatory proteins (G proteins) by Al3+, Mg2+, and F-. 19F NMR spectra of solutions containing Al3+, Mg2+, and F- showed a characteristic F- peak at -10 ppm. Addition of the GDP-bound form of either of two G protein alpha subunits (G alpha) resulted in the appearance of an additional peak at -29 or -30 ppm. This peak was not observed with guanosine 5'-3-O-(thio)triphosphate-G alpha or with GDP alone. Titration of Al3+, Mg2+, and F- indicated that each molecule of G alpha binds 3-5 molecules of F- (Kd = 0.47 mM), a single molecule of Al3+ (Kd much less than 0.1 mM), and a single Mg2+ ion (Kd about 0.1 mM). Replacement of Mg2+ with Mn2+ caused a dramatic broadening of the NMR signal, indicating that the metal ion binds in proximity to the protein-bound F- (less than 1 nm). 31P NMR of GDP-G alpha showed peaks at -2 and -8.6 ppm, corresponding to the beta- and alpha-phosphoryl groups of GDP, respectively. Binding of Al3+, Mg2+, and F- caused an upfield shift of 6 ppm for the beta-phosphoryl signal with no change in the alpha-phosphoryl signal. These observations indicate that Mg2+.GDP.AlF3-5 mimics Mg2+.GTP in its capacity to activate G protein alpha subunits.

PMID: 1899863 [PubMed - indexed for MEDLINE]


http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=1721077&dopt=Abstract

J Comp Neurol 1991 Oct 15;312(3):436-50

Developmental alterations in nociceptive threshold, immunoreactive calcitonin gene-related peptide and substance P, and fluoride-resistant acid phosphatase in neonatally capsaicin-treated rats.

Hammond DL, Ruda MA.

Department of Central Nervous System Diseases Research, G.D. Searle & Co., Skokie, Illinois 60077.

This study examined the effect of neonatal administration of capsaicin on nociceptive threshold and the distribution of calcitonin gene-related peptide (CGRP), substance P (SP), and fluoride-resistant acid phosphatase (FRAP) in the dorsal horn of the spinal cord during the course of development (10 days to 12 weeks of age) in the rat. As early as 10 days of age, CGRP-like immunoreactivity was reduced in laminae I, II, and V, as well as in the bundles of fibers situated dorsal and ventral to the central canal. However, beginning on or about 6 weeks of age, the density of CGRP-like immunoreactivity in the superficial laminae and in the bundles dorsal and ventral to the central canal increased. Moreover, thick, nonvaricose CGRP-like immunoreactive fibers appeared in laminae III and IV. These recurring fibers were of primary afferent origin as demonstrated by their disappearance after multiple, unilateral rhizotomies. A similar age-dependent alteration in the density of FRAP activity was also observed. Although virtually absent at 10 days of age after neonatal administration of capsaicin, the density of FRAP activity increased in lamina II by 8 weeks of age. This activity disappeared after multiple, unilateral rhizotomies, indicating that the FRAP activity that reappeared was of primary afferent origin. Neonatal administration of capsaicin also reduced the density of SP-like immunoreactivity in the dorsal horn as early as 10 days of age, although the density of SP-like immunoreactivity showed some recovery after 6 weeks of age. However, unlike CGRP-like immunoreactivity or FRAP activity, the density of SP-like immunoreactivity in capsaicin-treated rats was not detectably altered by multiple, unilateral rhizotomies, indicating that it originated principally from intrinsic dorsal horn neurons. Age-dependent alterations in both thermal and mechanical, but not chemical, nociceptive thresholds were also observed in these same animals. Thus, tail flick latency, hot plate latency, and paw withdrawal threshold were maximally increased at 6 weeks of age, after which time thresholds declined to vehicle-treated values. In contrast, capsaicin-treated animals were uniformly insensitive to ophthalmic administration of capsaicin. The correspondence between developmental alterations in CGRP-like immunoreactivity or FRAP activity and in thermal and mechanical nociceptive thresholds is suggestive of a role of CGRP- or FRAP-containing primary afferents in thermal and mechanical nociception.

PMID: 1721077 [PubMed - indexed for MEDLINE]


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