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Further evidence for synthesis of screening pigment granules involved in the photosensory membrane turnover of the crayfish photoreceptor.

Photosensory membrane degradation in crayfish occurs at first in multi-vesicular bodies (MVBs) and then, with the aid of lysosomal enzymes, in lysosome related lamellar bodies. In organ culture experiments with the isolated crayfish retina (Orconectes limosus) small screening pigment-like granules became visible under the electron microscope in such lamellar bodies and suggested a possible relation of photosensory membrane degradation and screening pigment granule synthesis. Chloroquine, an inhibitor of lysosomal activity, when added to the culture medium reduced the appearance of screening pigment-like granules in lamellar bodies, but led to the appearance of these granules in mature MVB's, indicating the involvement of lysosomal enzymes in the formation of pigmented lamellar bodies. In a second set of experiments the effect of bright light on the screening pigment granule ultrastructure of crayfish phoreceptors was investigated. It was found that after bright light exposure large numbers of little screening pigment granules (0.15-0.3 microns) were located between or close to rhabdomeral microvilli that were not at these sites in crayfish kept under natural light. MVB's were also reduced in size, and among the little screening pigmentary organelles granules of different electron density and morphology appeared. Additionally, vesicle flux to little screening pigment granules was detected. The screening pigment granules of the little type did not seem to be transported close to or between the microvilli, but appeared to be synthesized at these sites within little MVBs.

Animals↗

Cellular plasticity among axolotl neural crest-derived pigment cell lineages.

Many of the factors and mechanisms guiding the migration/differentiation of neural crest cells that give rise to a number of distinguishable cell types, including all dermal and epidermal pigment cells, remain unknown. The axolotl possesses three pigment cell types that differentiate according to specific developmentally programmed sequences and contribute to pigment pattern in the adult. A single lineage of the crest that becomes restricted to one of three pigment cell types gives us the opportunity to examine the existence of a neural crest stem cell population and the potential for trans-differentiation events. Interpretations of experiments involving drug-treated and mutant axolotls implicate cellular plasticity leading to observed phenotypes. We present results from recent in vitro studies designed to identify parameters influencing differentiation events of individual neural crest-derived pigment cell lineages. We demonstrate that the differentiation of xanthophores is enhanced, while that of the melanophores are inhibited in guanosine-supplemented neural crest cell cultures. Data suggest that the increase in one pigment cell population is at the expense of another, indicative of cellular plasticity. Videomicroscopy used in this study agrees with an abundance of correlative evidence supporting the hypothesis of transdifferentiation events among neural crest-derived pigment cell populations. The embryonic neural crest-derived pigment cell system is an ideal model to study differentiation of multipotential stem cells that play critical roles in patterning.

Ambystoma↗

The signaling pathway in photoresponses that may be mediated by visual pigments in erythrophores of Nile tilapia.

The ability to increase the synthesis or vary the distribution of pigment in response to light is an important feature of many pigment cells. Unlike other light-sensitive pigment cells, erythrophores of Nile tilapia change the direction of pigment migration depending on the peak wavelength of incident light: light near 365, 400 or 600 nm induces pigment aggregation, while dispersion occurs in response to light at 500 nm. How these phenomena are achieved is currently unknown. In the present study, the phototransduction involved in the pigment dispersion caused by light at 500 nm or the aggregation by light at 600 nm was examined, using pertussis toxin, cholera toxin, blockers of ion channels, various chemicals affecting serial steps of signaling pathways and membrane-permeable cAMP analog. The results show that light-induced bidirectional movements in tilapia erythrophores may be controlled by cytosolic cAMP levels via Gi- or Gs-type G proteins. In addition, RT-PCR demonstrated for the first time the expression of mRNAs encoding red and green opsins in tilapia fins, only where erythrophores exist. Here, we suggest that multiple cone-type visual pigments may be present in the erythrophores, and that unique cascades in which such opsins couple to Gi or Gs-type G proteins are involved in the photoresponses in these pigment cells. Thus, tilapia erythrophore system seems to be a nice model for understanding the photoresponses of cells other than visual cells.

Animals↗

In vivo and in vitro evidence of dermal fibroblasts influence on human epidermal pigmentation.

Using chimeric human epidermal reconstructs, we previously demonstrated that epidermal pigmentation is dependent upon the phototype of melanocytes. We report here several lines of experimental evidence for dermal modulation of human epidermal pigmentation. First, phototype II-III epidermal reconstructs grafted on the back of immunotolerant Swiss nu/nu mice developed a patchy pigmentation dependent on the presence of colonizing human or mouse fibroblasts. Similarly, human white Caucasoid split-thickness skin xenografted on the same mouse strain became black within 3 months and histochemistry revealed a phototype VI pattern of melanin distribution. In vitro, human fibroblasts colonizing human dead de-epidermized dermis (DDD) induced a decrease in epidermal pigmentation whereas mouse (Swiss nu/nu) fibroblasts increased epidermal pigmentation. Conditioned medium from mice (Swiss nu/nu) fibroblasts also increased pigmentation whereas conditioned medium from human fibroblasts had no significant effect. Lastly, epidermal reconstructs made with normal or vitiligo keratinocytes and/or normal or vitiligo melanocytes from the same donor grown on DDD originating from several donors of the same clinical phototype did not pigment similarly and no specific dermal influence was noted for vitiligo. Thus, fibroblast secretion and acellular dermal connective tissue itself significantly influence melanocyte proliferation and melanin distribution/degradation. Our study suggests that murine fibroblasts are more potent than human fibroblasts in secreting soluble factors which can act directly on pigmentation, such as SCF, or activate keratinocytes to produce basement membrane proteins or melanogenic factors.

Animals↗

Evaluation of the efficacy of the acellular dermal matrix allograft with partial thickness flap in the elimination of gingival melanin pigmentation. A comparative clinical study with 12 months of follow-up.

UNLABELLED: Acellular dermal matrix (ADM) has been used as a substitute for gingival allografts. The aim of this study was to evaluate the effectiveness of ADM with partial thickness flap in the elimination of gingival melanic pigmentations. Fifteen patients presenting bilateral gingival melanin pigmentation were selected for this study. Each side of the pigmentation was randomly assigned to be part of a group. The sites of the experimental group had a partial thickness flap raised, excised, followed by adaptation and suture of the ADM. On the opposite side (ie, sites from the control group), the oral epithelium was removed with a diamond bur. The healing process was evaluated at 1 and 2 weeks, and 1, 3, 6, 9, and 12 months postoperatively. Images of the patient's follow-up were digitalized, and occurrence and quantification of clinical repigmentation were evaluated. After 12 months, minimal repigmentation (mean 3.14 +/- 7.45%) was noted in 8 of 15 sites from the ADM group, while significant repigmentation (mean 55.84 +/- 27.25%) was seen in 15 of 15 abrasion sites. According to our results, it can be concluded that ADM may be successfully used in the elimination or greater reduction of gingival melanic pigmentations, and is more efficient than epithelium abrasion after 12 months (p < 0.0001). CLINICAL SIGNIFICANCE: Gingival pigmentation is a common finding in several ethnic groups. Although physiologic melanin pigmentation is not a medical problem, cosmetic demands may turn it into a problem of social acceptance in different cultures. In this way, different treatment modalities have been used with the aim of removing pigmentations for esthetic reasons. In the present study, the use of ADM with partial thickness flap resulted in more effective elimination of gingival melanin pigmentation, as repigmentation occurred later and was much weaker when compared to the outcomes of epithelial abrasion.

Adolescent↗

Pigmentation of the eye and hair in relation to age-related macular degeneration. An epidemiological study of 1000 aged individuals.

So far, no epidemiological studies have dealt with the relationship between dark and light pigmented Caucasians and the risk of developing age-related macular degeneration (AMD). Based upon hospital referred eye patients, dark ocular pigmentation has previously been suggested as a protective factor. The present study reviews macular degeneration, defined as macular changes implicating a vision of 6/9 or less, in prevalence rates in relation to ocular pigmentation and hair colour in an ethnic homogeneous and random population of 1000 Danes, 60-80 years of age. The population was predominantly light pigmented. Brown iris colour was only present in 15.9%, dark pigmented fundi in 19.0% and a black juvenile hair colour supposed in 6.5%. The prevalence rates of AMD ranged from 16.0 to 10.5% for brown respectively blue iris colour, and from 13.3% to 9.0% for dark respectively light pigmented fundi. The presented data indicate that no significant differences in prevalence rates of AMD seem to exist between dark and light ocular pigmented Danes. An additional comparison between 100 AMD patients referred to hospital and the epidemiological sample did not reveal a difference in pigmentation. The prevalence analysis also showed that neither is juvenile hair colour of prognostic value in terms of later occurrence of AMD nor seems greyness of hair to be an independent indicator of AMD, but related to age as is AMD, when simultaneously entered into a logistic regression model.

Aged↗

The gecko visual pigment: a pH indicator with a salt effect.

1. Unlike rhodopsin, the extracted 521-pigment of the Tokay gecko (Gekko gekko) is pH-sensitive and changes its spectral absorbance in the pH range of 4.5-7.3. The colour change is reversible and pH can be employed to adjust the spectral maximum anywhere between 490 nm and its native location at 521 nm.2. The hypsochromic shift with increasing acidity is opposite to that expected for the protonation of the Schiff base nitrogen and suggests an action on the secondary system of interacting charges that have long been postulated to adjust vertebrate visual pigment colour within the visible spectrum.3. Chloride ions modulate this pH effect in a systematic and significant manner. For the pigment extracted in the chloride-deficient state the colour change occurs in the pH range of 6.0-7.0, the midpoint being close to 6.5, suggesting the possible participation of the imidazole group of histidine as the functional moiety. With added NaCl the colour shifts to the region below pH 6.2.4. The modulating action of chloride is postulated to be a conformational change of the opsin leading to a shift of the secondary interacting site from one functional group to another or else to a change in pK of a single group due to the conformational alteration of the electrostatics of the system.5. At pH values between 7.5 and 9.0 a different mechanism becomes apparent. In this region a decrease occurs in the photopigment density as well as a shift in absorbance toward the blue. This alkaline effect is readily reversed either by adding NaCl or else by lowering the pH. Along with the other protective effects of chloride these ions serve to reduce or prevent this alkaline loss in density.6. Associated with this reversible photopigment loss is a reversible appearance of a product with a maximum at about 366 nm. The spectrum of this product is like that produced by the addition of 11-cis retinal to the extract. Acidification of the alkaline preparation leads to a restitution of the photopigment as well as to a reduction of the 366-product.7. Addition of hydroxylamine to the alkaline extract in appropriate concentration inhibits the restitution of pigment-521 with acid or NaCl, but adding 11-cis retinal to the system leads to restoration of the photopigment after acidification. All the evidence suggests that product-366 is either free 11-cis retinal or else held to the opsin in a form that does not alter its spectral absorbance. The alkaline effect is therefore a disruption of the aldimine bond of the visual pigment.8. In many respects the gecko 521-pigment behaves like the chicken cone pigment, iodopsin, suggesting that an investigation of the latter in terms of pH may be a worthy project for future study.9. With its ability to change colour with pH, with chloride, with nitrate, etc. the extractable gecko pigment offers possibilities for the investigation of mechanisms responsible for adjusting visual pigment absorbance throughout the visible spectrum. The techniques of circular dichroism, Raman spectroscopy, infra-red spectroscopy, etc. may find here a suitable material for these studies.

Animals↗

Rapid charge movements and photosensitivity of visual pigments in salamander rods and cones.

1. Photosensitivities of visual pigments were determined by measuring early receptor currents (ERCs) in voltage-clamped photoreceptors from larval salamanders. 2. As expected from previous work of others, the ERC elicited by a brief flash consisted of a rapid inward component followed by a larger and slower outward component. The magnitude of the outward component corresponded to the movement of about 0.18 electronic charge across the membrane per photoisomerization. 3. The time course of the ERC was independent of the flash intensity, the flash wavelength and the magnitude of the response. The outward component of the cone ERC declined about twice as rapidly as the outward component of the rod ERC.. 4. The amplitude of the ERC decreased as successive flashes bleached the cell's pigment. Using the proportional relation between the size of the ERC and the number of pigment molecules photoisomerized, photosensitivities of the native A2 pigments in rods, red-sensitive cones, blue-sensitive cones and UV-sensitive cones were determined. Calculated solution photosensitivities for rhodopsin, red-sensitive and blue-sensitive cone pigments were not significantly different and the average value for all three pigments at their respective absorption maxima was (7.3 +/- 1.6) x 10(-9) micron 2 molecule-1. A value of 44.0 x 10(-9) micron 2 molecule-1 was obtained in a single UV-sensitive cone. 5. Substitution of the native dehydroretinal chromophore in the red-sensitive cone pigment with 11-cis-retinal increased the solution photosensitivity to (9.6 +/- 0.62) x 10(-9) micron 2 molecule-1. 6. We conclude that cone pigments have large molecular absorption cross-sections and high quantum efficiencies of photoisomerization. These properties seem well suited for the receptive molecules of a highly sensitive, miniaturized transducer.

Animals↗

[Detection of new pigments related to purple urine bag syndrome].

It is well known that participation of two kinds of pigments, indigo blue and indigo red, are concerned with purple urine bag syndrome (PUBS). However, there is no research which describes the participation of other pigments. We separated three new kinds of red pigments, other than indigo blue and indigo red, from four patients with PUBS. One of the pigments was not visible to the naked eye and appeared only when an ultraviolet ray was irradiated. The appearance patterns of indigo blue, indigo red and the three new red pigments were in complete agreement in two cases, but differed in the other two cases. Moreover, bacteria isolated from the urine were cultured in an indican-added alkaline liquid media (pH9), 80% or more bacteria produced purple pigments. In case 1, from deep-colored pigments, each bacterium produced both indigo blue and indigo red, although new pigments could not be observed in the in vitro media.

Aged↗

Histoplasma capsulatum synthesizes melanin-like pigments in vitro and during mammalian infection.

Melanin is made by several important pathogenic fungi and has been implicated in the pathogenesis of a number of fungal infections. This study investigated whether the thermally dimorphic fungal pathogen Histoplasma capsulatum var. capsulatum produced melanin or melanin-like compounds in vitro and during infection. Growth of H. capsulatum mycelia in chemically defined minimal medium produced pigmented conidia. Growth of H. capsulatum yeast in chemically defined minimal medium with L-3,4-dihydroxyphenylalanine (DOPA) or (-)-epinephrine produced pigmented cells. Treatment of the pigmented cells with proteolytic enzymes, denaturant, and hot concentrated acid yielded dark particles that were similar in size and shape to their respective propagules. Melanin-binding monoclonal antibodies (MAb) labeled pigmented conidia, yeast, and the isolated particles as determined by immunofluorescence microscopy. Electron spin resonance spectroscopy revealed that pigmented yeast cells and particles derived from pigmented cells were stable free radicals consistent with their identification as melanins. Tissues from mice infected with H. capsulatum and from biopsy specimens from a patient with histoplasmosis contained fungal cells that were labeled by melanin-binding MAb. Digestion of infected mouse tissues yielded dark particles that reacted with the melanin-binding MAb and were similar in appearance to H. capsulatum yeast cells. Additionally, sera from infected mice contained antibodies that bound melanin particles. Phenoloxidase activity capable of synthesizing melanin from L-DOPA was detected in cytoplasmic yeast cell extracts. These findings indicate that H. capsulatum conidia and yeast can produce melanin or melanin-like compounds in vitro and that yeast cells can synthesize pigment in vivo. Since melanin is an important virulence factor in other pathogenic fungi, this pigment may have a similar role to play in the pathogenesis of histoplasmosis.

Animals↗

A Streptomyces avermitilis gene encoding a 4-hydroxyphenylpyruvic acid dioxygenase-like protein that directs the production of homogentisic acid and an ochronotic pigment in Escherichia coli.

A 1.5-kb genomic fragment isolated from Streptomyces avermitilis that directs the synthesis of a brown pigment in Escherichia coli was characterized. Since pigment production in recombinant E. coli was enhanced by the addition of tyrosine to the medium, it had been inferred that the cloned DNA might be associated with melanin biosynthesis. Hybridization studies, however, showed that the pigment gene isolated from S. avermitilis was unrelated to the Streptomyces antibioticus melC2 determinant, which is the prototype of melanin genes in Streptomyces spp. Sequence analysis of the 1.5-kb DNA that caused pigment production revealed a single open reading frame encoding a protein of 41.6 kDa (380 amino acids) that resembled several prokaryotic and eukaryotic 4-hydroxyphenylpyruvate dioxygenases (HPDs). When this open reading frame was overexpressed in E. coli, a protein of about 41 kDa was detected. This E. coli clone produced homogentisic acid (HGA), which is the expected product of the oxidation of 4-hydroxyphenylpyruvate catalyzed by an HPD, and also a brown pigment with characteristics similar to the pigment observed in the urine of alkaptonuric patients. Alkaptonuria is a genetic disease in which inability to metabolize HGA leads to increasing concentrations of this acid in urine, followed by oxidation and polymerization of HGA to an ochronotic pigment. Similarly, the production of ochronotic-like pigment in the recombinant E. coli clone overexpressing the S. avermitilis gene encoding HPD is likely to be due to the spontaneous oxidation and polymerization of the HGA accumulated in the medium by this clone.

4-Hydroxyphenylpyruvate Dioxygenase↗

Pigment production by Cryptococcus neoformans and other Cryptococcus species from aminophenols and diaminobenzenes.

Cryptococcus neoformans and other Cryptococcus species can produce pigment(s) from many aminophenol and diaminobenzene compounds. Pigment production from these compounds is similar to the conversion of diphenols to melanin by C. neoformans. Several pigmentation patterns (resulting in the identification or grouping of Cryptococcus species) have been observed by using diaminobenzene and aminophenol compounds as substrates. The most common pigmentation pattern observed was pigment production by both C. neoformans and C. terreus. In contrast to the diphenols, only two aminophenols (4-hydroxymetanilamide and 3-aminotyrosine) were found to be highly specific as substrates. They allowed only C. neoformans to produce pigment. When 4-aminosalicylic acid was the substrate, a unique pattern was observed because only C. terreus, C. diffluens, and C. albidus produced pigment. Finally, a pattern was observed in which C. neoformans produced large amounts of pigment from aminophenol and diaminobenzene compounds, whereas the other Cryptococcus species produced smaller amounts. A simplified scheme with three substrates resulted in the identification of C. terreus and C. neoformans as well as two groups of other Cryptococcus species, group I (C. albidus and C. diffluens) and group II (C. laurentii and C. luteolus).

Aminobenzoates↗

Production by Bacillis subtilis of brown sporulation-associated pigments.

The mode of production of the brown pigments of Bacillus subtilis 168 L-4, pigments frequently used as phenotypic markers for sporulation in this organism, has been studied. A defined liquid medium which promoted maximal pigment formation was developed. Five brown components, which could be resolved by thin-layer chromatography, were produced in the culture broth. Removal of cells from the medium at the end of logarithmic growth did not alter the type or amount of the pigments formed, indicating that the cells excreted pigment precursors into the medium during growth. Pigment formation from the precursors was found to occur by an oxygen-requiring, base-dependent, Mn2+-requiring, nonenzymatic pathway. Pigment production was also stimulated by the presence of tyrosine and histidine in the medium. The increases in extracellular pH often associated with spore formation in B. subtilis might be the cause of the concomitant appearance of brown pigments.

Bacillus subtilis↗

Fibroblast growth factors are necessary for neural retina but not pigmented epithelium differentiation in chick embryos.

During eye development, optic vesicles evaginate laterally from the neural tube and develop into two bilayered eye cups that are composed of an outer pigment epithelium layer and an inner neural retina layer. Despite their similar embryonic origin, the pigment epithelium and neural retina differentiate into two very distinct tissues. Previous studies have demonstrated that the developmental potential of the pigmented epithelial cells is not completely restricted; until embryonic day 4.5 in chick embryos, the cells are able to switch their phenotype and differentiate into neural retina when treated with fibroblast growth factors (FGF) (Park, C. M., and Hollenberg, M. J. (1989). Dev. Biol. 134, 201-205; Pittack, C., Jones, M., and Reh, T. A. 1991). Development 113, 577-588; Guillemot, F. and Cepko, C. L. (1992). Development 114, 743-754). These studies motivated us to test whether FGF is necessary for neural retina differentiation during the initial stages of eye cup development. Optic vesicles from embryonic day 1.5 chick were cultured for 24 hours as explants in the presence of FGF or neutralizing antibodies to FGF2. The cultured optic vesicles formed eye cups that contained a lens vesicle, neural retina and pigmented epithelium, based on morphology and expression of neural and pigmented epithelium-specific antigens. Addition of FGF to the optic vesicles caused the presumptive pigmented epithelium to undergo neuronal differentiation and, as a consequence, a double retina was formed. By contrast, neutralizing antibodies to FGF2 blocked neural differentiation in the presumptive neural retina, without affecting pigmented epithelial cell differentiation. These data, along with evidence for expression of several FGF family members and their receptors in the developing eye, indicate that members of the FGF family may be required for establishing the distinction between the neural retina and pigmented epithelium in the optic vesicle.

Animals↗

Microspectrophotometry of visual pigments and oil droplets in a marine bird, the wedge-tailed shearwater Puffinus pacificus: topographic variations in photoreceptor spectral characteristics.

Microspectrophotometric examination of the retina of a procellariiform marine bird, the wedge-tailed shearwater Puffinus pacificus, revealed the presence of five different types of vitamin A(1)-based visual pigment in seven different types of photoreceptor. A single class of rod contained a medium-wavelength sensitive visual pigment with a wavelength of maximum absorbance (lambda(max)) at 502 nm. Four different types of single cone contained visual pigments maximally sensitive in either the violet (VS, lambda(max) 406 nm), short (SWS, lambda(max) 450 nm), medium (MWS, lambda(max) 503 nm) or long (LWS, lambda(max) 566 nm) spectral ranges. In the peripheral retina, the SWS, MWS and LWS single cones contained pigmented oil droplets in their inner segments with cut-off wavelengths (lambda(cut)) at 445 (C-type), 506 (Y-type) and 562 nm (R-type), respectively. The VS visual pigment was paired with a transparent (T-type) oil droplet that displayed no significant absorption above at least 370 nm. Both the principal and accessory members of the double cone pair contained the same 566 nm lambda(max) visual pigment as the LWS single cones but only the principal member contained an oil droplet, which had a lambda(cut) at 413 nm. The retina had a horizontal band or 'visual streak' of increased photoreceptor density running across the retina approximately 1.5 mm dorsal to the top of the pecten. Cones in the centre of the horizontal streak were smaller and had oil droplets that were either transparent/colourless or much less pigmented than at the periphery. It is proposed that the reduction in cone oil droplet pigmentation in retinal areas associated with high visual acuity is an adaptation to compensate for the reduced photon capture ability of the narrower photoreceptors found there. Measurements of the spectral transmittance of the ocular media reveal that wavelengths down to at least 300 nm would be transmitted to the retina.

Animals↗

Genetic basis of spectral tuning in the violet-sensitive visual pigment of African clawed frog, Xenopus laevis.

Ultraviolet (UV) and violet vision in vertebrates is mediated by UV and violet visual pigments that absorb light maximally (lambdamax) at approximately 360 and 390-440 nm, respectively. So far, a total of 11 amino acid sites only in transmembrane (TM) helices I-III are known to be involved in the functional differentiation of these short wavelength-sensitive type 1 (SWS1) pigments. Here, we have constructed chimeric pigments between the violet pigment of African clawed frog (Xenopus laevis) and its ancestral UV pigment. The results show that not only are the absorption spectra of these pigments modulated strongly by amino acids in TM I-VII, but also, for unknown reasons, the overall effect of amino acid changes in TM IV-VII on the lambdamax-shift is abolished. The spectral tuning of the contemporary frog pigment is explained by amino acid replacements F86M, V91I, T93P, V109A, E113D, L116V, and S118T, in which V91I and V109A are previously unknown, increasing the total number of critical amino acid sites that are involved in the spectral tuning of SWS1 pigments in vertebrates to 13.

Amino Acid Substitution↗

Observations on pigment granules in the bones of silky fowls.

The distribution of melanin pigment-containing cells in the bones of both young and adult silky fowls was observed. Melanin pigment was detected not only in melanocytes which were mainly distributed in the periosteum, but also in all the other types of cells in the periosteum and bone. The continuity of the number of pigment granules in melanocytes and that in the other pigment-containing cells could not be recognized because the granules in the latter cells were much fewer than those in the former. In young fowls, the pigment-containing cells were distributed in all layers of the periosteum and bone, but their number was low. On the other hand, in aged fowls, most of the cells in the periosteum had pigment granules. In the bone, however, pigment granules were observed only in osteocyte situated near the surface. These findings suggest that the pigment granules which are observed in osteocytes have been transferred from melanocytes to osteogenic cells or osteoblasts before they differentiate to osteocytes, where they are presumed to be digested.

Animals↗

Tea pigments induce cell-cycle arrest and apoptosis in HepG2 cells.

AIM: To investigate the molecular mechanisms by which tea pigments exert preventive effects on liver carcinogenesis. METHODS: HepG2 cells were seeded at a density of 5X10(5)/well in six-well culture dishes and incubated overnight. The cells then were treated with various concentrations of tea pigments over 3 d, harvested by trypsinization, and counted using a hemocytometer. Flow cytometric analysis was performed by a flow cytometer after propidium iodide labeling. Bcl-2 and p21(WAF1) proteins were determined by Western blotting. In addition, DNA laddering assay was performed on treated and untreated cultured HepG2 cells. RESULTS: Tea pigments inhibited the growth of HepG2 cells in a dose-dependent manner. Flow-cytometric analysis showed that tea pigments arrested cell cycle progression at G1 phase. DNA laddering was used to investigate apoptotic cell death, and the result showed that 100 mg/L of tea pigments caused typical DNA laddering. Our study also showed that tea pigments induced upregulation of p21(WAF1) protein and downregulation of Bcl-2 protein. CONCLUSION: Tea pigments induce cell-cycle arrest and apoptosis. Tea pigments may be used as an ideal chemopreventive agent.

Anticarcinogenic Agents↗