Mark D. Rollag

Melatonin-Depleted Blood from Premenopausal Women Exposed to Light at Night Stimulates Growth of Human Breast Cancer Xenografts in Nude Rats

The increased breast cancer risk in female night shift workers has been postulated to result from the suppression of pineal melatonin production by exposure to light at night. Exposure of rats bearing rat hepatomas or human breast cancer xenografts to increasing intensities of white fluorescent light during each 12-hour dark phase (0-345 microW/cm2) resulted in a dose-dependent suppression of nocturnal melatonin blood levels and a stimulation of tumor growth and linoleic acid uptake/metabolism to the mitogenic molecule 13-hydroxyoctadecadienoic acid. Venous blood samples were collected from healthy, premenopausal female volunteers during either the daytime, nighttime, or nighttime following 90 minutes of ocular bright, white fluorescent light exposure at 580 microW/cm2 (i.e., 2,800 lx). Compared with tumors perfused with daytime-collected melatonin-deficient blood, human breast cancer xenografts and rat hepatomas perfused in situ, with nocturnal, physiologically melatonin-rich blood collected during the night, exhibited markedly suppressed proliferative activity and linoleic acid uptake/metabolism. Tumors perfused with melatonin-deficient blood collected following ocular exposure to light at night exhibited the daytime pattern of high tumor proliferative activity. These results are the first to show that the tumor growth response to exposure to light during darkness is intensity dependent and that the human nocturnal, circadian melatonin signal not only inhibits human breast cancer growth but that this effect is extinguished by short-term ocular exposure to bright, white light at night. These mechanistic studies are the first to provide a rational biological explanation for the increased breast cancer risk in female night shift workers.

Photic regulation of melatonin in humans: ocular and neural signal transduction.

Light is a potent stimulus for regulating the pineal glands production of melatonin and the broader circadian system in humans. It initially was thought that only very bright photic stimuli (≥ 2500 lux) could suppress nocturnal melatonin secretion and induce other circadian responses. It is now known that markedly lower illuminances (≤ 200 lux) can acutely suppress melatonin or entrain and phase shift melatonin rhythms when exposure conditions are optimized. The elements for physical/biological stimulus processing that regulate photic influences on melatonin secretion include the physics of the light source, gaze behavior relative to the light source, and the transduction of light energy through the pupil and ocular media. Elements for sensory/neural signal processing become involved as photons are absorbed by retinal photopigments and neural signals are generated in the retinohypothalamic tract. Aspects of this physiology include the ability of the circadian system to integrate photic stimuli spatially and temporally as well as the wavelength sensitivity of the operative photoreceptors. Acute, light-induced suppression of melatonin is proving to be a powerful tool for clarifying how these elements of ocular and neural physiology influence the interaction between light and the secretion of melatonin from the human pineal gland.

Role of a pineal cAMP-operated arylalkylamine N-acetyltransferase/14-3-3-binding switch in melatonin synthesis.

The daily rhythm in melatonin levels is controlled by cAMP through actions on the penultimate enzyme in melatonin synthesis, arylalkylamine N-acetyltransferase (AANAT; serotonin N-acetyltransferase, EC 2.3.1.87). Results presented here describe a regulatory/binding sequence in AANAT that encodes a cAMP-operated binding switch through which cAMP-regulated protein kinase-catalyzed phosphorylation [RRHTLPAN → RRHpTLPAN] promotes formation of a complex with 14-3-3 proteins. Formation of this AANAT/14-3-3 complex enhances melatonin production by shielding AANAT from dephosphorylation and/or proteolysis and by decreasing the Km for 5-hydroxytryptamine (serotonin). Similar switches could play a role in cAMP signal transduction in other biological systems.

Sensitivity of the Human Circadian System to Short-Wavelength (420-nm) Light

The circadian and neurobehavioral effects of light are primarily mediated by a retinal ganglion cell photoreceptor in the mammalian eye containing the photopigment melanopsin. Nine action spectrum studies using rodents, monkeys, and humans for these responses indicate peak sensitivities in the blue region of the visible spectrum ranging from 459 to 484 nm, with some disagreement in short-wavelength sensitivity of the spectrum. The aim of this work was to quantify the sensitivity of human volunteers to monochromatic 420-nm light for plasma melatonin suppression. Adult female (n = 14) and male (n = 12) subjects participated in 2 studies, each employing a within-subjects design. In a fluence-response study, subjects (n = 8) were tested with 8 light irradiances at 420 nm ranging over a 4-log unit photon density range of 1010 to 1014 photons/cm 2/sec and 1 dark exposure control night. In the other study, subjects (n = 18) completed an experiment comparing melatonin suppression with equal photon doses (1.21 × 1013 photons/cm2/sec) of 420 nm and 460 nm monochromatic light and a dark exposure control night. The first study demonstrated a clear fluence-response relationship between 420-nm light and melatonin suppression (p < 0.001) with a half-saturation constant of 2.74 × 1011 photons/cm2/sec. The second study showed that 460-nm light is significantly stronger than 420-nm light for suppressing melatonin (p < 0.04). Together, the results clarify the visible short-wavelength sensitivity of the human melatonin suppression action spectrum. This basic physiological finding may be useful for optimizing lighting for therapeutic and other applications.

Blue light from light-emitting diodes elicits a dose-dependent suppression of melatonin in humans

Light suppresses melatonin in humans, with the strongest response occurring in the short-wavelength portion of the spectrum between 446 and 477 nm that appears blue. Blue monochromatic light has also been shown to be more effective than longer-wavelength light for enhancing alertness. Disturbed circadian rhythms and sleep loss have been described as risk factors for astronauts and NASA ground control workers, as well as civilians. Such disturbances can result in impaired alertness and diminished performance. Prior to exposing subjects to short-wavelength light from light-emitting diodes (LEDs) (peak λ = 469 nm; 1/2 peak bandwidth = 26 nm), the ocular safety exposure to the blue LED light was confirmed by an independent hazard analysis using the American Conference of Governmental Industrial Hygienists exposure limits. Subsequently, a fluence-response curve was developed for plasma melatonin suppression in healthy subjects (n = 8; mean age of 23.9 ± 0.5 years) exposed to a range of irradiances of blue LED light. Subjects with freely reactive pupils were exposed to light between 2:00 and 3:30 AM. Blood samples were collected before and after light exposures and quantified for melatonin. The results demonstrate that increasing irradiances of narrowband blue-appearing light can elicit increasing plasma melatonin suppression in healthy subjects (P < 0.0001). The data were fit to a sigmoidal fluence-response curve (R(2) = 0.99; ED(50) = 14.19 μW/cm(2)). A comparison of mean melatonin suppression with 40 μW/cm(2) from 4,000 K broadband white fluorescent light, currently used in most general lighting fixtures, suggests that narrow bandwidth blue LED light may be stronger than 4,000 K white fluorescent light for suppressing melatonin.

Melanopsin, Ganglion-Cell Photoreceptors, and Mammalian Photoentrainment

An understanding of the retinal mechanisms in mammalian photoentrainment will greatly facilitate optimization of the wavelength, intensity, and duration of phototherapeutic treatments designed to phase shift endogenous biological rhythms. A small population of widely dispersed retinal ganglion cells projecting to the suprachiasmatic nucleus in the hypothalamus is the source of the critical photic input. Recent evidence has shown that many of these ganglion cells are directly photosensitive and serve as photoreceptors. Melanopsin, a presumptive photopigment, is an essential component in the phototransduction cascade within these intrinsically photosensitive ganglion cells and plays an important role in the retinal photoentrainment pathway. This review summarizes recent findings related to melanopsin and melanopsin ganglion cells and lists other retinal proteins that might serve as photopigments in the mammalian photoentrainment input pathway.

Pertussis toxin blocks melatonin-induced pigment aggregation inXenopus dermal melanophores

SummaryThe molecular mechanism of action for the pineal hormone melatonin was explored by testing melatonin interaction with the components of the hormone-sensitive adenylate cyclase complex in aXenopus dermal melanophore bioassay. Forskolin was employed to stimulate melanosome dispersion. The ability of melatonin to reverse forskolin-stimulated pigment dispersion was assessed, as was the effect of pertussis toxin on the ability of melatonin to aggregate dispersed pigment.Forskolin elicited dispersal of melanosomes in a dose dependent manner (EC50=12 nM) in meninges from stage 52–56 tadpoles ofXenopus laevis. Maximal pigment dispersion was obtained with 100 nM forskolin. Melatonin reversed this effect of forskolin (EC50=1.5 nM), causing pigment aggregation. Pertussis toxin blocked the melatonin-induced aggregation (EC50=358 ng/ml).Prior treatment of the melanophore containing meningeal explants with pertussis toxin results in blockade of melatonin induced pigment aggregation. A 41 kDa pertussis toxin substrate is found in explant homogenates treated with32P-NAD and pertussis toxin. The availability of this substrate is reduced by prior treatment of intact explants with pertussis toxin and depletion of melatonin responsiveness corresponds to depletion of the 41 kDa substrate. Together, these data suggest that melatonin action upon amphibian dermal melanosomes is mediated by a system requiring a protein similar to the regulatory protein Ni used by mammalian cells to mediate the action of hormones which inhibit adenylate cyclase through a cell surface receptor.

Ultraviolet regulation of neuroendocrine and circadian physiology in rodents

UV wavelengths can regulate neuroendocrine and circadian responses in some rodent species. Appropriately timed UV exposures can block the short photoperiod-induced collapse of the reproductive system, cause a rapid suppression of nocturnal melatonin synthesis, regulate melatonin rhythms and phase shift wheel running rhythms. These biological effects of UV are not dependent on the Harderian gland or melanin in the eye, but appear to be related to the degree of transmission through the ocular lens. Such results are consistent with the hypothesis that elements in the retina can transduce UV stimuli for circadian and neuroendocrine regulation.

Dim Light Adaptation Attenuates Acute Melatonin Suppression in Humans

Abstract Studies in rodents with retinal degeneration indicated that neither the rod nor the cone photoreceptors obligatorily participate in circadian responses to light, including melatonin suppression and photoperiodic response. Yet there is a residual phase-shifting response in melanopsin knockout mice, which suggests an alternate or redundant means for light input to the SCN of the hypothalamus. The findings of Aggelopoulos and Meissl suggest a complex, dynamic interrelationship between the classic visual photoreceptors and SCN cell sensitivity to light stimuli, relative to various adaptive lighting conditions. These studies raised the possibility that the phototransductive physiology of the retinohypothalamic tract in humans might be modulated by the visual rod and cone photoreceptors. The aim of the following two-part study was to test the hypothesis that dim light adaptation will dampen the subsequent suppression of melatonin by monochromatic light in healthy human subjects. Each experiment included 5 female and 3 male human subjects between the ages of 18 and 30 years, with normal color vision. Dim white light and darkness adaptation exposures occurred between midnight and 0200 h, and a full-field 460-nm light exposure subsequently occurred between 0200 and 0330-h for each adaptation condition, at 2 different intensities. Plasma samples were drawn following the 2-h adaptation, as well as after the 460-nm monochromatic light exposure, and melatonin was measured by radioimmunoassay. Comparison of melatonin suppression responses to monochromatic light in both studies revealed a loss of significant suppression after dim white light adaptation compared with dark adaptation (p < 0.04 and p < 0.01). These findings indicate that the activity of the novel circadian photoreceptive system in humans is subject to subthreshold modulation of its sensitivity to subsequent monochromatic light exposure, varying with the conditions of light adaptation prior to exposure.

Suppression of pineal melatonin in Peromyscus leucopus by different monochromatic wavelengths of visible and near-ultraviolet light (UV-A).

The purpose of this study was to examine the effects of monochromatic visible and near-ultraviolet radiation (UV-A) on pineal melatonin suppression in the white-footed mouse, Peromyscus leucopus. To this end, mice were entrained to a daily cycle of 8 h of light and 16 h of darkness. During the night when pineal melatonin contents were high, mice were individually exposed for 5 min to specific wavelengths of monochromatic light (10 nm half-peak bandwidths). Control animals received the same handling conditions but no experimental exposure. Pineal glands were collected from animals 18 min after the 5 min experimental exposure and were later assayed for melatonin content. In groups of animals exposed to equal photon densities (2.64 X 10(15) photons/cm2) of either 320, 340, 360, 500, or 560 nm, mean pineal melatonin content was significantly suppressed as compared to the unexposed control animals. The 640 nm wavelength (red) at the same photon density did not suppress pineal melatonin. These experiments are the first to demonstrate light-induced suppression of pineal melatonin in Peromyscus leucopus. In addition, these data reveal a novel finding: the suppression of pineal melatonin content by ultraviolet wavelengths as low as 320 and 340 nm.