Biological activity of Melaleuca alternifola (Tea Tree) oil component, terpinen-4-ol, in human myelocytic cell line HL-60.

BACKGROUND: Tea tree oil is an aboriginal Australian traditional medicine for bruises, insect bites, and skin infections. It was rediscovered in the 1920s as a topical antiseptic that is more effective than Phenol. Previous studies have demonstrated its antiseptic qualities, but its effects on human white blood cells have never been investigated. OBJECTIVE: To test the hypothesis that tea tree oil exerts its antiseptic action through white blood cell activation. METHODS: Crude oil and the purified "active" component were studied by using a model system that responds to bioactive components by induction of differentiation in white blood cells. Methods used included white blood cell oxidative burst assay (nitroblue tetrazolium [NBT] dye reduction); cell proliferation assay (tritiated thymidine incorporation); cell surface differentiation marker assay (flow cytometric quantitation of phycoerythrin-anti-CD 11b binding); cell viability assay (trypan blue exclusion); and cellular differentiation enzyme assay (white cell esterase staining). RESULTS: Collectively, five assays that measure differentiation in white blood cells indicated monocytic differentiation after treatment with either crude oil or the purified active component. Both the crude oil and the purified active component, (+:-) terpinene-4-ol, caused a similar type and amount of differentiation. The culture of cells in medium containing serum caused more activation than in medium containing no serum. CONCLUSION: The antiseptic activity of tea tree oil appears to be due, in part, to white blood cell activation.

Basic science research in chiropractic: the state of the art and recommendations for a research agenda.

A position paper was prepared as background information for participants in the National Workshop to Develop the Chiropractic Workshop Agenda conducted by the Palmer Center for Chiropractic Research, Davenport, Iowa. The paper was revised in light of comments and suggestions at the workshop by participants and by a workgroup composed of basic scientists from within and outside of chiropractic. This final article documents the history of basic science research in chiropractic, and the current state of the art of basic science research conducted since 1975 by chiropractors or investigators at chiropractic institutions in North America. Seed recommendations contained in the working paper for the development of an adequate infrastructure needed to conduct future research and seed recommendations for a future basic science research agenda were also modified and revised by the workgroup participants through a modified nominal group process. The final recommendations contained in this article represent a synthesis of these recommendations and those of the authors.

Mutations in the c-erbA beta 1 gene: do they underlie euthyroid fibromyalgia?

Fibromyalgia, a chronic condition of widespread pain, stiffness, and fatigue, has proven unresponsive to drugs, the use of which is based on the 'serotonin-deficiency hypothesis'. An alternative hypothesis-failed transcription regulation by thyroid hormone-can explain the serotonin deficiency and other objective findings and symptoms of euthyroid fibromyalgia. Virtually every feature of fibromyalgia corresponds to signs or symptoms associated with failed transcription regulation by thyroid hormone. In hypothyroid fibromyalgia, failed transcription regulation would result from thyroid-hormone deficiency. In euthyroid fibromyalgia, failed transcription regulation may result from low-affinity thyroid hormone receptors coded by a mutated c-erbA beta 1 gene, yielding partial peripheral resistance to thyroid hormone. The hypothesis of this paper is that, in euthyroid fibromyalgia, a mutant c-erbA beta 1 gene (or alternately, the c-erbA alpha 1 gene) results in low-affinity thyroid-hormone receptors that prevent normal thyroid hormone regulation of transcription. As in hypothyroidism, this would cause a shift toward alpha-adrenergic dominance and increases in both cyclic adenosine 3'-5'-phosphate phosphodiesterase and inhibitory Gi proteins. The result would be tissue-specific hypothyroid-like symptoms despite normal circulating thyroid-hormone levels.

Glucuronidation of all-trans-retinoic acid in liposomal membranes.

Retinoyl beta-D-glucuronide is a biologically active metabolite of retinoic acid. The kinetics of UDP-glucuronosyltransferase-catalyzed biosynthesis of retinoyl beta-D-glucuronide was examined in rat liver and intestinal native microsomes incubated with [3H retinoic acid incorporated into liposomes. The product was identified by cochromatography with authentic all-trans retinoyl beta-D-glucuronide, by hydrolysis with beta-D-glucuronidase, and by mass spectrometry. In vitamin A-sufficient rats the apparent Km values for all-trans-retinoic acid were 173 microM and 125 microM, and the apparent Vmax, 62 and 41 pmol/min per mg, for small intestinal and liver microsomes, respectively. In vitamin A-deficient rats repleted with all-trans-retinyl acetate, the apparent Km (91 microM) and Vmax (53 pmol/min per mg) for intestinal microsomes were in range of those of vitamin A-sufficient rats. The similarities in the kinetic parameters for UDP-glucuronosyltransferase in small intestinal mucosa and liver suggest that the reactions are catalyzed by the same enzyme. In vitamin A-deficient rats given a large amount all-trans-retinoic acid (1.2 mmol/day for 3 days) the apparent Km was 105 microM and Vmax, 127 pmol/min per mg of intestinal microsomal protein. We conclude that the kinetics of intestinal retinoic acid glucuronidation are not characteristic of simple detoxification reactions. Retinoyl glucuronide may be important in mediating retinoic acid metabolism and function.

Effect of hexachlorobiphenyl on vitamin A homeostasis in the rat.

Vitamin A status and turnover were examined in rats that had been exposed to chronic dietary treatment of 3,4,5,3',4',5'-hexachlorobiphenyl (HCB), 1 mg/kg diet. HCB caused hepatic depletion and renal accumulation of vitamin A, and a 1.7-fold increase in the serum retinol concentration. Intravenously administered [3H]retinol bound to retinol binding protein-transthyretin complex (RBP-TTR complex) was used to study the dynamics of circulatory retinol in these rats. In HCB-treated rats, the plasma turnover rate of retinol was increased compared to vitamin A-adequate untreated controls. HCB caused a 50% reduction of total radioactivity in liver, and, except for 0.5 h after the [3H]retinol-RBP-TTR dose, the specific activity of the hepatic retinyl ester pool was greater compared to control rats. The kidneys of HCB-treated rats accumulated radioactivity in the retinyl ester fraction. HCB also caused a 50% reduction in adrenal radioactivity compared with control rats. Urinary and fecal excretion of radioactivity was 3-fold higher in HCB-treated rats as compared to controls. Our findings demonstrate that chronic HCB feeding results in expansion of plasma vitamin A mass, in changes of liver and kidney retinol and retinyl ester pool dynamics and in an increased metabolism of vitamin A.

Vitamin A metabolism in rats chronically treated with 3,3',4,4',5,5'-hexabromobiphenyl.

Chronic dietary administration of 3,3',4,4',5,5'-hexabromobiphenyl (HBB), 1 mg/kg diet, caused a decrease in retinol (20-fold) and retinyl esters (23-fold) in the livers of female rats, but resulted in a 6.4-fold increase in retinol and 7.4-fold increase in retinyl esters in the kidneys. Liver acyl-CoA:retinol acyltransferase and retinyl palmitate hydrolase activities were reduced while serum concentration of retinol was unaffected by HBB feeding. Metabolism of a physiological dose of [11-3H]retinyl acetate (10 micrograms), was examined in rats fed either vitamin A-adequate diet, or marginal amounts of vitamin A, or vitamin A-adequate diet containing HBB. A 13-fold greater amount of the administered vitamin A was found in kidneys of HBB-treated rats. In rats fed adequate or low amounts of vitamin A, kidney radioactivity was primarily in the retinol fraction, while in HBB-fed rats the radioactivity was associated mostly with retinyl esters. Fecal and urinary excretion of radioactivity was greatly increased in HBB-treated rats. Chronic HBB feeding results in a loss of ability of liver to store vitamin A, and severely alters the uptake and metabolism of vitamin A in the kidneys. We conclude that HBB causes major disturbances in the regulation of vitamin A metabolism.

Induction of differentiation of human promyelocytic leukemia cell line HL-60 by retinoyl glucuronide, a biologically active metabolite of vitamin A.

We examined the differentiation activity of retinoyl beta-D-glucuronide, a biologically active physiological metabolite of vitamin A, using the human promyelocytic leukemic cell line HL-60, which can be induced to differentiate with retinoic acid. Retinoyl beta-D-glucuronide (1 microM) inhibited HL-60 cell proliferation by 55-75%, inhibited tritiated thymidine incorporation into DNA by 63-80%, and induced 38-50% of the cells to differentiate into mature granulocytes. The potency of growth inhibition and induction of differentiation by retinoyl beta-D-glucuronide was similar to that of all-trans-retinoic acid. The continuous presence of either retinoyl beta-D-glucuronide or all-trans-retinoic acid was not required to obtain maximum growth arrest and differentiation: a 1-hr exposure of HL-60 cells to the retinoids gave the same response (measured after a total incubation time of 48 hr) as a 24-hr or 48-hr continuous treatment. Retinoyl beta-D-glucuronide (0.1-0.2 mM) was 50% less cytotoxic to HL-60 cells than all-trans-retinoic acid at an equimolar concentration. Retinoyl beta-D-glucuronide was not significantly metabolized to other retinoids; retinoic acid was not formed during incubation. We conclude that retinoyl beta-D-glucuronide can arrest HL-60 cell proliferation and induce their differentiation into mature granulocytes; it may act by itself or by being hydrolyzed to retinoic acid, which could be immediately utilized and metabolized. The therapeutic use of this retinoid as an antineoplastic agent is suggested.

Effect of moderate vitamin A supplementation and lack of dietary vitamin A on the development of mammary tumors in female rats treated with low carcinogenic dose levels of 7,12-dimethylbenz(a)anthracene.

We examined the effect of moderately increased and of marginal continued dietary supplementation of vitamin A (retinyl acetate) and the effect of lack of dietary vitamin A on the initiation and promotion stages of mammary tumorigenesis in female Sprague-Dawley rats treated with a single low (0.5 mg/100 g body weight) or very low (0.1 mg/100 g body weight) dose of i.v.-administered 7,12-dimethylbenz(a)anthracene. The number of mammary tumors was significantly (P less than 0.05) reduced if prior to and during initiation with 7,12-dimethylbenz(a)anthracene the rats were fed a moderately increased (30 micrograms/day) or marginal (3 micrograms/day) amount of vitamin A, compared to rats fed an adequate (10 micrograms/day) amount of vitamin A. The number of mammary tumors was also significantly (P less than 0.05) reduced when a moderately increased or marginal amount of vitamin A was provided during the tumor promotion phase. In addition, the number of mammary tumors was significantly (P less than 0.05) reduced by the lack of dietary vitamin A during both the initiation and promotion stages of this tumorigenic process, when compared to vitamin A adequate, ad libitum-fed rats, but not when compared to vitamin A adequate, food-restricted controls. The reduction in numbers of mammary tumors observed in these studies was reflected primarily in significant (P less than 0.05) decreases in mammary fibroadenomas; the number of mammary carcinomas was often reduced, but due to a low frequency of the carcinomatous lesions, this reduction did not reach the 5% level of statistical probability. Plasma and liver vitamin A levels were determined during both the initiation and promotion stages. As the dietary supplementation of vitamin A increased from 0 to 30 micrograms/day, there was an increase in mean liver and plasma vitamin A levels. No consistent correlation between plasma and liver vitamin A levels and the occurrence of mammary tumors was observed, except with the moderately increased (30 micrograms/day) intake of vitamin A, that resulted in a small, but statistically significant (P less than 0.05) increase of serum retinol at initiation; this may account for the observed reduction in mammary tumors. These results provide evidence that moderate alterations in vitamin A consumption can modulate low-dose chemically induced mammary gland tumorigenesis. Most importantly, suppression of mammary gland tumorigenesis can be achieved by moderately increased, frequent, and regular consumption of vitamin A; prolonged consumption of vitamin A-deficient diets or diets marginal in vitamin A does not enhance the risk of mammary tumor development.

Quantitation of biological retinoids by high-pressure liquid chromatography: primary internal standardization using tritiated retinoids.

A single method is described for quantitation of 14 retinoids found in biological material. The method consists of reversed-phase HPLC, internal standardization, and carrier extraction procedures with three synthetic retinoids. Primary standardization of HPLC uv detector is achieved using tritiated all-trans-retinoic acid, all-trans-retinol, all-trans-retinyl palmitate, and all-trans-retinyl acetate. Extraction methods are standardized by correlating the uv absorbance of retinoids at 340 nm with radioactivity of tritiated retinoids of known specific activity. Quantitation of 10 pg of tritiated or 5 ng of nonradioactive retinoid per 0.1 g sample in a polarity range from 4-oxo-retinoic acid to retinyl stearate can be achieved in a single, 50-min chromatographic run. A single HPLC pump, a C18 reversed-phase analytical column, a multistep three-solvent gradient, and inexpensive solvents based on methanol, water, and chloroform comprise this cost-effective chromatographic system. Our primary standardization method allows investigators employing different procedures to compare results between laboratories by standardizing the HPLC uv detector with commercially available tritiated retinoids. With this method we were able to quantitate nanomolar amounts of endogenous retinoic acids and retinyl esters, that "HPLC uv only" conditions usually would not detect in the circulation and liver of rats under physiological conditions.

Influence of dietary retinyl acetate on normal rat mammary gland development and on the enhancement of 7,12-dimethylbenz[a]anthracene-induced rat mammary tumorigenesis by high levels of dietary fat.

The effect of high levels of dietary fat and retinyl acetate (ROA) on 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat mammary tumor development and growth was examined. Female Sprague-Dawley rats, 51-53 days of age, were treated ig with 5 mg DMBA. At 55-57 days of age, the animals were divided into the following dietary treatment groups: A) 4.5% fat [control fat (CF)]; B) CF + 1.0 mmol ROA/kg diet (CF + ROA); C) 20.0% fat [high fat (HF)]; and D) HF + ROA. HF diets significantly increased mammary tumor multiplicity, with or without ROA, but did not significantly influence mammary tumor growth. ROA treatment reduced mammary tumor multiplicity regardless of the level of dietary fat and inhibited mammary tumor growth in the presence of normal levels of dietary fat. High levels of dietary fat did not significantly influence normal mammary gland growth and development. ROA significantly decreased normal mammary gland growth and development regardless of the level of dietary fat. Blood retinoids in rats fed ROA were primarily in the form of retinyl esters, i.e., retinyl linoleate, retinyl palmitate-oleate, and retinyl stearate. Free retinol levels in blood were not significantly influenced by ROA feeding. Blood retinyl ester levels were lower in rats fed the HF + ROA diet as compared to rats fed the CF + ROA diet.

Acute polybrominated biphenyl toxicosis alters vitamin A homeostasis and enhances degradation of vitamin A.

A single oral dose of HBB (3,3',4,4',5,5',-hexabromobiphenyl), a congener of the polybrominated biphenyls (PBBs), and a dioxin-type cytochrome P-450 enzyme inducer rapidly and dramatically altered the steady-state metabolism of vitamin A and caused an abnormal twofold enhancement in the metabolic output of degraded vitamin A in urine and feces of rats. The effects are most likely explained by an increased metabolism of vitamin A in kidney and deregulation of vitamin A metabolism in liver; this may lead to an increased dietary vitamin A requirement.

Metabolism of all-trans-retinoic acid and all-trans-retinyl acetate. Demonstration of common physiological metabolites in rat small intestinal mucosa and circulation.

The kinetics and metabolism of physiological doses of all-trans-retinoic acid were examined in blood and small intestinal mucosa of vitamin A-depleted rats. A major portion of intrajugularly injected retinoic acid is rapidly (within 2 min) sequestered by tissues; subsequently 13-cis-retinoic acid and polar metabolites are released into circulation. All-trans-retinoic acid appears in small intestinal epithelium within 2 min after dosing and is the major radioactive compound there for at least 2 h. Retinoyl glucuronide and 13-cis-retinoic acid are early metabolites of all-trans-retinoic acid in the small intestine of bile duct-cannulated rats. Retinoyl glucuronide, the major metabolite of retinoic acid intestinal epithelium, in contrast to other polar metabolites, was not detected in circulation. An examination of [3H]retinyl acetate metabolites under steady state conditions in vitamin A-repleted rats demonstrates the occurrence of all-trans-retinoic acid and 13-cis-retinoic acid in circulation and in intestinal epithelium, in a pattern similar to that found after injection of retinoic acid into vitamin A-depleted rats. Our data establish that all-trans-retinoic acid, 13-cis-retinoic acid, and retinoyl glucuronide are physiological metabolites of vitamin A in target tissues, and therefore are important candidates as mediators of the biological effect of the vitamin.

Comparison of fetal and adult retinol and retinoic acid binding proteins in bovine serum and pigment epithelium.

Cellular retinol binding protein (CRBP) and cellular retinoic acid binding protein (CRABP) were assayed in bovine eye pigment epithelium (PE) cytosol from both early fetal and young adult animals. Both fetal and adult PE contain CRBP but only fetal PE contains CRABP. Blood retinol binding protein (RBP) was also assayed in serum from fetal and adult animals. Both fetal and adult serum contain RBP. A novel albumin adsorption technique (Affi-Gel Blue) combined with refinements of the sucrose gradient centrifugation technique provide a better assay method for retinoid receptors than polyacrylamide disc gel electrophoresis. Our findings support the hypothesis that retinoid receptors may cause or be the consequence of differentiation in animal tissues.

Analysis of retinol and dideuterated retinol in rat plasma by gas chromatography combined mass spectrometry.

An isotope dilution method for determining the total body stores of vitamin A using a hydrocarbon derivative of rat plasma retinol, anhydroretinol (ANR) or dideuteroanhydroretinol (2H2ANR) has been evaluated by gas chromatography combined mass spectrometry (GC/MS). Rats with negligible vitamin A stores were dosed with retinyl acetate (ROA) or ROA and 11,12dideuteroretinyl acetate (2H2ROA). The 2H2ROA dose was allowed to equilibrate with total body stores for seven days and the deuterium/hydrogen ratio (D/H ratio) of rat plasma retinol fraction was determined. The results indicate that after the equilibration period the plasma D/H ratio is 73 to 108 percent of the loading dose D/H ratio. This study extends earlier reports that exogenous vitamin A storage is lower than the expected fifty percent figure in rats with low initial total body stores of vitamin A [1, 7, 8, 15, 17]. This work supports the concept that the estimation of total body stores of vitamin A by an isotope dilution method is useful for populations with negligible stores (i.e. liver stores) of vitamin A.

Analysis of deuterated analogs of vitamin A by electron impact and chemical ionization modes in gas chromatography coupled to mass spectrometry.

To evaluate the total body reserves of vitamin A in humans, deuterated forms of vitamin A are potentially useful probes. In the present investigation, we have selected anhydroretinol as a useful indicator of retinol isolated from plasma during GC/MS analysis because of its relatively high volatility, its formation from retinol in good yield directly on GC columns, and the improbability of deuterium loss or exchange during its formation. The major drawback in its use is the extensive isomerization to cis-isomers which occurred on GC columns even under mild conditions of analysis. Favorable conditions for the GC/MS assay of anhydroretinol in the EI mode were defined. The mass spectral response is linear with the amount of retinol injected from 10 to 400 ng, and the observed sensitivity is adequate for the measurement of retinol in 1 ml of plasma. By using isobutane or methane, chemical ionization mass spectra of anhydroretinol and retinaldehyde are reported for the first time. Although both gave the expected [M + H]+ molecular ion, analysis of anhydroretinol in the EI mode was a more appropriate and sensitive measure of retinol under our assay conditions.

Population density and regulation of cell division in 3T3 cells. I. Inorganic phosphate levels, uptake and release.

Triggering mechanisms for initiating density dependent inhibition of cell division in 3T3 cell monolayers are activated approximately two to three population doublings prior to cessation of cell division at monolayer confluency. This activation occurs at a critical contact cell density of approximately 8 X 10(3) cells/cm2. During this period there are selective controls on transport and storage of required low molecular weight nutrients. A possible correlation between orthophosphate and rates of cell division has been investigated. We have demonstrated a relationship between cellular concentrations of orthophosphate and initiation of density dependent inhibition of cell division. Prior to critical intercellular contact, the [Pi] in 3T3 is 10 mM. During critical contact, this concentration is quickly reduced to approximately 2 mM and remains at this concentration to confluency. Similar alterations do not occur in Py 3T3 cells, which maintain a concentration of approximately 2 mM Pi regardless of cell density. After confluent 3T3 cells are released from inhibition of cell division the [Pi] must increase several-fold before DNA synthesis commences. These are physiological changes in 3T3 cellular [Pi] as a function of cell density, and cannot be attributed to nutrient depletion, altered transport of Pi into the cell, increased [ATP], or increased [PPi] levels. The controlled modulation of [Pi] may regulate glycolysis and coordinate counter-ion changes (Ca++) may regulate mitochondrial activity.