Photoperiod modulates the inhibitory effect of in vitro melatonin on lymphocyte proliferation in female Siberian hamsters.

In Siberian hamsters (Phodopus sungorus), short days suppress reproductive function and lymphocyte proliferation. To determine whether melatonin influences cell-mediated immunity through a direct action on lymphocyte proliferation, in vitro responsiveness to mitogens and melatonin was assessed in systemic and splenic lymphocytes from adult female Siberian hamsters housed in either long or short days for 13 weeks. Short days provoked reproductive regression and reduced lymphocyte proliferation. Physiological concentrations of melatonin (50 pg/ml) inhibited in vitro proliferation of circulating lymphocytes, whereas higher concentrations (> or = 500 pg/ml) were required to inhibit proliferation of splenic lymphocytes. Immunomodulatory effects of melatonin were restricted to lymphocytes from long-day hamsters-in vitro melatonin had no effect on circulating or splenic lymphocytes from females in short days. Responsiveness to melatonin in short-day lymphocytes may be restrained by the already expanded nightly pattern of melatonin secretion in short days. These data support the hypothesis that melatonin acts directly on lymphocytes from long-day hamsters to suppress blastogenesis.     

Melatonin directly stimulates the secretion of progesterone by human and bovine granulosa cells in vitro

Melatonin, at concentrations and periods of exposure reflecting those present during the circadian cycle, was investigated for its influence on steroid production by granulosa cells cultured in serum-supplemented medium. At high (200 pg/ml) but not low (20 pg/ml) physiological concentrations, melatonin significantly stimulated progesterone production by human granulosa cells. This response was independent of the overall level of cell activity and was seen under the different culture conditions associated with different culture media. Exposure to melatonin for 8 h significantly stimulated progesterone secretion to a level similar to that achieved under continuous exposure, and the effect was reduced to control levels during subsequent periods in which no melatonin was added. Melatonin had no consistent effect on aromatase activity in the conversion of stored or serum-available androgen to oestradiol. Melatonin significantly stimulated progesterone production by bovine granulosa cells in vitro, at concentrations similar to those present during the endogenous nocturnal rise (100-400 pg/ml). This response to physiological conditions by human and bovine cells suggests a role for melatonin in the regulation of progesterone production by the ovary.     

Induction of lymphokine-activated killer cells from rat thymocytes using recombinant human interleukin-2

Summary Using a 4-h ⁵¹Cr release assay, we observed that thymocytes from Fischer strain rats incubated with recombinant human interleukin-2 (rhIL-2) developed cytotoxicity to YAC-1 lymphoma, 9L-glioma, and B-16 melanoma cells (effector/target ratio =25/1). Induction of the lymphokine-activated killer (LAK) cells was as follows: (1) when 5×10⁶/ml thymocytes were cultured with various concentrations of rhIL-2 (50, 125, 250, 500, or 1,000 units/ml) for 4 days, no cell proliferation was observed at any concentration. However, the LAK cells showed significant cytotoxicity toward all tumor cells at more than 50 units/ml. (2) When 5×10⁶/ml thymocytes were cultured for 1 to 6 days with 250 units/ml of rhIL-2, the harvested cell count decreased markedly after the 2nd day. The cytotoxicity of all the tumor cells became significant after the 2nd day, with peak activity on the 4th day. In rat splenocytes, on the other hand, the LAK cells could not be identified because rat splenocytes developed nonspecific cytotoxicity in medium containing fetal calf serum without adding rhIL-2.     

Effect of different fetal bovine serum concentrations on the replicative life span of cultured chick cells

Summary The effect of Eagle's minimal essential medium, containing different fetal bovine serum (FBS) concentrations, on the proliferation and replicative life span of cultured chick cells has been studied. Our results showed that the rate of chick cell proliferation and the cell density at stationary phase increased as a function of serum concentration between 5 and 30% FBS. The replicative life span of cultured chick cells was dependent on the FBS concentration between 5 and 20% in a medium volume of 0.20 ml/cm². The maximum replicative life span of chick cells was obtained by serially propagating cells in a medium volume of 0.20 ml/cm² containing 20 or 30% FBS, or, alternatively, in 0.53 ml/cm² containing 10, 20 or 30% FBS. Cells grown in medium containing 5% serum had a calendar life span of 35 days, whereas cells propagated in medium containing higher serum concentrations had a calendar life span of 50 days. These results reenforce the concept that, although the kinetics of cell population aging can be affected by the culture medium composition, the aging of cells in culture is controlled by alterations within the cell. This work was supported by IIT Research Institute.     

Production of human epidermal growth factor by an ampicillin resistant recombinant Escherichia coli strain

Summary The effect of medium composition and initial glucose concentration on production of hEGF by recombinant E. coli cells was investigated. Optimum hEGF production was observed in a yeast extract/acid hydrolysed casein/salts media containing an initial glucose concentration of 10 g.l^-1. A maximum hEGF titer of 250 mg.l^-1 was obtained in this medium after 32 h in laboratory fermenters with pH, temperature, agitation and aeration set at 6.8, 30°C, 500 rpm and 2 vvm, respectively.     

Human epidermal growth factor and the proliferation of human fibroblasts

The effect of human epidermal growth factor (hEGF), a 5,400 molecular weight polypeptide isolated from human urine, on the growth of human foreskin fibroblasts (HF cells) was studied by measuring cell numbers and the incorporation of labeled thymidine. The addition of hEGF to HF cells growing in a medium containing 10% calf serum resulted in a 4-fold increase in the final density. The presence of hEGF also promoted the growth of HF cells in media containing either 1% calf serum or 10% gamma globulin-free serum. The addition of hEGF to quiescent confluent monolayers of HF cells, maintained in a medium with 1% calf serum for 48 hours, resulted in a 10- to 20-fold increase in the amount of ³H-thymidine incorporation after 20–24 hours. The stimulation of thymidine incorporation was maximal at an hEGF concentration of 2 ng/ml, was dependent on the presence of serum, and was enhanced by the addition of ascorbic acid. In confluent cultures of HF cells, subject to density dependent inhibition of growth, hEGF was able to stimulate DNA synthesis more effectively than fresh calf serum. Human EGF stimulated DNA synthesis in quiescent cultures, however, regardless of cell density. The addition of rabbit anti-hEGF inhibited all effects of this growth factor on HF cells.     

Involvement of steroid hormones on in vitro maturation of pig oocytes

The purpose of this study was to determine if the addition of steroid hormones into the culture medium could influence the in vitro maturation of pig oocytes. The cumulus-oocyte complexes (COCs). collected from follicles of 2-5 mm diameter, were matured in steroid-free medium supplemented with various concentrations of estradiol-17beta (0-3000 ng/ml), progesterone (0-5000 ng/ml) and testosterone (0-300 ng/ml). The COCs were cultured for 42 h, then fertilized in vitro. We analyzed nuclear and cytoplasmic maturation with lacmoid stain 20 h after in vitro insemination. We observed no significant effect (P > 0.05) on the percentage of oocytes completing nuclear or cytoplasmic maturation or the number of sperm penetrating each oocyte for any concentration of progesterone, estradiol-17beta or testosterone. Similarly, adding a combination of those hormones to the medium did not significantly (P > 0.05) affect any of the criteria. In order to determine if there was a possible secretion of steroids during maturation, we added COCs, denuded oocytes and stripped cumulus cells to drops of a steroid-free medium and cultured them for 42 h, after which we analyzed the medium, before and after culture, for the presence of progesterone, estradiol-17beta and testosterone by radioimmunoassay (RIA) analysis. COCs, as well as cumulus cells alone, secreted similar amounts of estradiol (43.3 and 37.5 pg/ml, respectively) and progesterone (4.24 and 4.79 ng/ml, respectively) into the maturation medium. A small amount of estradiol (28.8 pg/ml) was also detected when oocytes were cultured alone. These results indicate that no steroids need to be added to the maturation medium of pig oocytes and that the COCs secrete steroids during maturation. It is possible that the amounts produced by the COCs fulfill any requirement for steroids if these steroids are required for either nuclear or cytoplasmic oocyte maturation.     

Microcarrier culture of recombinant Chinese hamster ovary cells for production of human immune interferon and human tissue-type plasminogen activator

Summary Recombinant Chinese hamster ovary cells were successfully cultured semi-continuously on microcarriers of gelatin or modified dextran under non-selective conditions for up to three weeks. High and constant production rates for human immune interferon and tissue-type plasminogen activator were obtained. For cells that produced interferon, the highest cell concentration and interferon production was obtained with gelatin microcarriers though the specific production when grown in the presence of 0.2% fetal calf serum was slightly higher for cells cultured on dextran microcarriers (0.12 U/cell day versus 0.11 U/cell day). For cells that produced plasminogen activator, a slightly higher cell concentration was obtained for cells grown on dextran microcarriers (9x10⁵ cells/ml versus 7x10⁵ cells/ml). However, the specific and total production rates were significantly higher for cells cultured on gelatin microcarriers (6.7 pg/cell day versus 2.1 pg/cell day). The maximum cell concentration and specific production rate could be increased to 2.3x10⁶ cells/ml and 3.4 pg/cell day for dextran microcarriers by adding 6-aminohexanoic acid to the medium. For gelatin microcarriers, the addition of 6-aminohexanoic acid increased the specific production rate to 14.4 pg/cell day. Cell growth, however, was inhibited.     

Circadian rhythms of melatonin release from chicken pineal in vitro: modified melatonin radioimmunoassay

An improved and simplified radioimmunoassay for measuring pineal, serum, and in vitro cultured medium melatonin is described. Using 2-[125I]iodomelatonin as radiolabeled ligand and a polyclonal rabbit antimelatonin antiserum, melatonin concentrations were determined in all three types of samples by a 2-day direct equilibrium double-antibody assay method without prior extraction. Serial dilutions of pineal homogenates, serum, and cultured medium all gave parallel displacement curves. Cross-reactivity of the antisera with other indoles was negligible. Intraassay coefficients of variation (n = 3) were 5.09, 3.32, and 5.05% at 7.81, 62.5, and 500 pg/tube, respectively, and the interassay coefficients of variation (n = 20) were 12.18% at 62.5 pg/tube. A characteristic diurnal rhythm of melatonin was observed using this direct assay for measuring daytime and nighttime chicken pineal and serum samples. An in vitro incubation of chicken pineal glands with a lighting cycle of 12-hr light:12-hr dark showed that the diurnal rhythm of melatonin secretion into the cultured medium was maintained. The direct assay method described in this report for measuring chicken melatonin using 2-[125I]iodomelatonin as radiolabeled ligand coupled with the in vitro cultured chicken pineal gland clearly offers great potential for studying the chicken pineal circadian oscillator and its underlying mechanism.     

Melatonin increases p53 and p21WAF1 expression in MCF-7 human breast cancer cells in vitro.

The aim of the present work was to study whether melatonin, at physiological concentrations, exerts its antiproliferative effects on MCF-7 human breast cancer cells by inducing the expression of some of the proteins involved in the control of the cell cycle. MCF-7 cells were cultured for 48 h in DMEM media containing either melatonin (1 nM) or the diluent (0.001% ethanol). At this concentration, after 48 hours of incubation, melatonin reduced the number of viable cells in relation to controls. The decreased cell proliferation was coincident with a significant increase in the expression of p53 as well as p21WAF1 proteins. These results demonstrate that melatonin inhibits MCF-7 cell proliferation by inducing an arrest of cell cycle dependent on an increased expression of p21WAF1 protein, which is mediated by the p53 pathway.     

Dose-dependent disruptive effects of melatonin,light and temperature on regeneration in the planarian Phagocata gracilis

Many critical cellular processes, including cell proliferation and tissue repair, exhibit melatonin sensitivity. We examined the effects of exogenous melatonin, photoperiod and temperature on regeneration in Phagocata gracilis, a common North American turbellarian. Planarian regeneration involves the proliferation and migration of pluripotent stem cells capable of replacing missing body components. A characteristic mass of cells (blastema) forms during planarian regeneration, and its formation, growth and differentiation provide reliable endpoints for the study of regenerative processes. We tested the effects of exogenous melatonin at two concentrations and three photoperiods on both head and tail regenerates under seasonal diurnal temperature regimes. High temperatures and melatonin treatment increased mortality in P. gracilis, and surviving melatonin-treated subjects exhibited delayed regeneration. Asymmetrical and deficient regeneration was more common in melatonin-treated, high temperature and dark treated subjects. Additionally, high temperature and melatonin induced a novel locomotor dysfunction.     

Antioxidant activity of melatonin in Chinese hamster ovarian cells: changes in cellular proliferation and differentiation

Melatonin is an endogenously generated molecule with free radical scavenging and antioxidant properties. Here, we studied the antiproliferative role of melatonin and other antioxidants on transformed Chinese hamster ovarian cells. Melatonin reduces cell proliferation in a dose- and time-dependent manner. Natural antioxidants which appear in edible plants including resveratrol and vitamin E mimicked the effect of melatonin. Flow cytometer analysis revealed that melatonin treatment reduces the number of cells in S-phase and increases cells in both G0/G1 and G2/M gaps. In addition, melatonin, as well as trolox, caused a clear morphological change by inducing the cells to become spindle shaped and fibroblast-like. Its effect is a reversible phenomenon that disappeared when melatonin was withdrawn from the culture medium. GSH levels are increased after melatonin treatment but pharmacologically blockade of GSH synthesis did not abolish melatonin's antiproliferative effect. Reduction of cell proliferation and the apparent induction of cell differentiation overlapped with melatonin's ability to change the intracellular redox state of CHO cells. We conclude that the cellular redox state may be involved in cellular transformation caused by antioxidants such as melatonin and trolox.     

The response of human peripheral blood lymphocytes to phytohemagglutinin: determination of cell numbers.

Optimization and definition of conditions for studying lymphocyte function in vitro resulted in exponential proliferation of lymphocytes from day 2 to day 5 with an average doubling time of 20 hr. The number of cells in culture on day 5 was 5–10 times as great as the number initially planted and 10–20 times as great as the number surviving in culture on day 2. An improved pronase-cetrimide technique was used to determine the number of viable lymphocytes as a function of time after addition of PHA. The volume changes in nuclei, obtained after cetrimide treatment, were quantitated using a curve-fitting computer program.The response could be described in terms of an induction phase (0–2 days) characterized by a decrease in cellularity and an increase in nuclear volume, a proliferation phase (2–5 days) characterized by an exponential proliferation and a continued increase in the number of cells having a large nuclear volume, and a lysis phase (5–14 days) characterized by a decrease in cellularity and a decrease in nuclear volume. The results reported here suggest that the ratio of the number of cells cultured to the volume of culture medium was crucial for optimal transformation and proliferation, 10⁵ cells/ml producing far better responses than 10⁶ cells/ml.     

CXCL8 and CCL20 Enhance Osteoclastogenesis via Modulation of Cytokine Production by Human Primary Osteoblasts

Generalized osteoporosis is common in patients with inflammatory diseases, possibly because of circulating inflammatory factors that affect osteoblast and osteoclast formation and activity. Serum levels of the inflammatory factors CXCL8 and CCL20 are elevated in rheumatoid arthritis, but whether these factors affect bone metabolism is unknown. We hypothesized that CXCL8 and CCL20 decrease osteoblast proliferation and differentiation, and enhance osteoblast-mediated osteoclast formation and activity. Human primary osteoblasts were cultured with or without CXCL8 (2–200 pg/ml) or CCL20 (5–500 pg/ml) for 14 days. Osteoblast proliferation and gene expression of matrix proteins and cytokines were analyzed. Osteoclast precursors were cultured with CXCL8 (200 pg/ml) and CCL20 (500 pg/ml), or with conditioned medium (CM) from CXCL8 and CCL20-treated osteoblasts with or without IL-6 inhibitor. After 3 weeks osteoclast formation and activity were determined. CXCL8 (200 pg/ml) and CCL20 (500 pg/ml) enhanced mRNA expression of KI67 (2.5–2.7-fold), ALP (1.6–1.7-fold), and IL-6 protein production (1.3–1.6-fold) by osteoblasts. CXCL8-CM enhanced the number of osteoclasts with 3–5 nuclei (1.7-fold), and with >5 nuclei (3-fold). CCL20-CM enhanced the number of osteoclasts with 3–5 nuclei (1.3-fold), and with >5 nuclei (2.8-fold). IL-6 inhibition reduced the stimulatory effect of CXCL8-CM and CCL20-CM on formation of osteoclasts. In conclusion, CXCL8 and CCL20 did not decrease osteoblast proliferation or gene expression of matrix proteins. CXCL8 and CCL20 did not directly affect osteoclastogenesis. However, CXCL8 and CCL20 enhanced osteoblast-mediated osteoclastogenesis, partly via IL-6 production, suggesting that CXCL8 and CCL20 may contribute to osteoporosis in rheumatoid arthritis by affecting bone cell communication.     

Effect of biosynthetic human epidermal growth factor on the synthesis and secretion of mucin glycoprotein from primary culture of rabbit fundal mucosa cells

Summary Biosynthetic human epidermal growth factor (Bh-EGF) induced dose-dependent synthesis and secretion of neutral mucin glycoprotein when the fundal cells isolated from rabbit stomach were cultured in serum-free medium containing Bh-EGF at concentrations as high as 10 to 100 ng/ml. At these high concentrations, Bh-EGF had no effect on the cell growth. In marked contrast, much lower concentrations from 0.1 to 1.0 ng/ml of Bh-EGF failed to stimulate mucin synthesis, but enhanced proliferation of the cells. Electrophoretic pattern of the mucin secreted from the cultured mucosal cells was very similar to that of the authentic mucin obtained from rabbit stomach. Maximal secretion of the mucin from the cells was observed at Hour 96 of the culture. Although fetal bovine serum (5%) and insulin (0.5 μg/ml) also stimulated the mucosal cells, both in growth and in mucin synthesis and release, the enhancing activity of the mucin synthesized and released by Bh-EGF at a concentration of 100 ng/ml per microgram DNA of cultured cells was far superior to that of 5% fetal bovine serum and 0.5 μg/ml insulin.     

Pyruvate-Induced changes in hepatoma cell morphology and macromolecular synthesis

Cells maintained in basal growth medium with 0.2–1.0% serum often require citric acid cycle intermediates for optimal viability. We have found that pyruvate added to minimal growth medium causes cellular flattening and formation of external processes accompanied by increaded DNA synthesis in cultured hepatoma cells (HTC cells). Cells were cultured in plalstic T-flasks (0.5, 1.0, or 2.0 × 10⁶ cells/flask) containing 5 ml medium (90% Eagle's Basal Medium (BME) and 10% Swim's S-77) with various concentrations of fetal calf serum (0.2,0.25, 0.5, 1.0, 2.0, 10%) and either pyruvate (50, 100, 250,500, 1,000μg/ml), or one of: dibutyryl cAMP (DBcAMP) or dibutyryl cGMP (DBcGMP) at 10^?3, 10^?4, or 10^?5 M. At 44–48 hr cultures were pulsed with tritiated thymidine, uridine, or lecucine. Cells became attached to the plastic surface within 24hr. Cells in medium with 0.25 to 2.0% serum had a rounded appearance. With added pyruvate, cellular flattening, process formation, and an increased adherence to the substratum was absorbed. By 48 hr, culture without pyruvate grew in rounded clusters; with pyruvate, cells formed extensive interconnecting processes that appeared loosely attached to the monolayer surface. At the cell densities tested, process formation was maximal with 250 to 500 μg/ml pyruvate. Cytochalasin B blocked flattening and process formation; EDTA (1 mg/ml) caused retraction of processes within 3 min, and a slow dissolution of these structures within cells was observed. DBcAMP or DBcGMP did not induce process formation. Flattening and process foormation in pyruvate-enriched cultures were accompanied by marked stimulation of DNA synthesis and smaller increases in RNA and protein synthesis. Cell number was not affected. These pyruvate-induced changes suggest that alterations in energy metabolism, or precursors that enhance viability and macromolecular synthesis in mammalian cell cultures, may exert marked effects on cellular morphology without corresponding changes in growth of neoplastic liver cells.     

Selective and efficient culturing of retinal pigment epithelial cells using a feeder layer

BACKGROUND: The techniques to isolate and purify retinal pigment epithelial (RPE) cells from small piece of autologous tissues are extremely difficult, and it is important to develop an efficient cell culture technique for RPE cells. The purpose of this study was to investigate the effect of 3T3-J2 cells and conditioned medium from 3T3-J2 cells on the proliferation of cultured RPE cells. METHODS: RPE cells from pigmented rabbits and a human RPE-derived cell line, ARPE-19, were used. First, the effects of co-culturing RPE cells with 3T3-J2 cells on the growth of the cells were analyzed. Second, the effects of the conditioned medium from 3T3-J2 cells on the proliferation of both types of cells were investigated. And third, the effects of the conditioned medium on RPE cell culture from a surgically removed choroidal neovascular (CNV) membrane were investigated. RESULTS: The 3T3-J2 cells increased the proliferation of both rabbit RPE cells and ARPE-19 cells. The number of rabbit RPE cells cultured in a mixture of the conditioned medium from 3T3-J2 cells was significantly higher than that in the reported optimal condition, and a similar tendency was observed for ARPE-19 cells. The results from enzyme-linked immunosorbent assay showed the presence of PDGF-AB, VEGF and IGF-I in the conditioned medium. The conditioned medium also promoted selective growth of human RPE cells from CNV. DISCUSSION: The results from this study present the conditions for efficient and selective culture of primary RPE cells.     

Heparin inhibits proliferation of fetal vascular smooth muscle cells in the absence of platelet-derived growth factor

Proliferation of smooth muscle cells from the pulmonary arteries and aortas of fetal calves is inhibited by heparin in vitro. This effect is reversible and dose dependent. Comparisons with effects of other polysaccharides indicate that only extensively sulfated polysaccharides inhibit proliferation of smooth muscle cells but that specific structural features of heparin are required to achieve maximum effect. Heparin-Sepharose chromatography of medium containing fetal calf serum reduces the ability of that medium to promote growth of smooth muscle cells from fetal pulmonary arteries, suggesting that heparin may remove soluble growth factors in serum. However, inhibition of fetal pulmonary artery smooth muscle cell proliferation by heparin is identical in media supplemented either with serum prepared from fetal calf plasma, in which platelet-derived growth factor (PDGF) is not detectable, or with fetal calf serum, which contains relatively abundant PDGF (114 pg/ml). Thus, inhibition of fetal pulmonary artery smooth muscle cell proliferation by heparin is not mediated solely by decreased availability or activity of exogenous PDGF. These studies suggest that morphogenesis of the smooth muscle investment of the pulmonary arteries could be regulated by local production of heparin-like inhibitors of smooth muscle cell growth.     

Ammonia effects in cultures of normal and transformed 3T3 cells

3T3 and SV-40 transformed 3T3 mouse fibroblasts were cultured in media with serum and antibiotics plus ammonia (NH₃ z NH₄⁺) added as NH₄C1. Both cell lines cultured without added ammonia showed normal morphology and multiplication even though ammonia in the medium at the end of the culture period ranged from 35 to 48 μg/ml. Ammonia concentrations being significantly higher in media removed from cells at the end of the culture period than in media incubated identically without cells, verified that cells released substantial quantities of ammonia in addition to components of the medium which underwent spontaneous breakdown. Both cell lines showed changes in morphology and highly significant reductions in cell multiplication which increased progressively as the concentration of added ammonia on the initial day of culture was increased to 35μg/ml. Control 3T3 cultures released significantly greater quantities of ammonia per cell than control cultures of transformed cells but their multiplication was more adversely affected by added ammonia. There were downward shifts in pH of the culturing medium for both cell lines as culture age increased at all concentrations of added ammonia, However, significant reductions in cell multiplication resulted from additions of ammonia that did not produce significant changes in extracellular pH. The data show that studies upon the effects of pH of the medium on cultured cells require control of ammonia concentrations.     

Influence of Light Intensity on Plasma Melatonin and Locomotor Activity Rhythms in Tench

Melatonin production by the pineal organ is influenced by light intensity, as has been described in most vertebrate species, in which melatonin is considered a synchronizer of circadian rhythms. In tench, strict nocturnal activity rhythms have been described, although the role of melatonin has not been clarified. In this study we investigated daily activity and melatonin rhythms under 12∶12 light‐dark (LD) conditions with two different light intensities (58.6 and 1,091 µW/cm²), and the effect of 1 h broad spectrum white light pulses of different intensities (3.3, 5.3, 10.5, 1,091.4 µW/cm²) applied at middarkness (MD) on nocturnal circulating melatonin. The results showed that plasma melatonin in tench under LD 12∶12 and high light conditions displayed rhythmic variation, where values at MD (255.8±65.9 pg/ml) were higher than at midlight (ML) (70.7±31.9 pg/ml). Such a difference between MD and ML values was reduced in animals exposed to LD 12∶12 and low light intensity. The application of 1 h light pulses at MD lowered plasma melatonin to 111.6±3.2 pg/ml (in the 3.3–10.5 µW/cm² range) and to 61.8±18.3 pg/ml (with the 1,091.4 µW/cm² light pulse) and totally suppressed nocturnal locomotor activity. These results show that melatonin rhythms persisted in tench exposed to low light intensity although the amplitude of the rhythm is affected. In addition, it was observed that light pulses applied at MD affected plasma melatonin content and locomotor activity. Such a low threshold suggests that the melatonin system is capable of transducing light even under dim conditions, which may be used by this nocturnal fish to synchronize to weak night light signals (e.g., moonlight cycles).