Disposition of glycolic acid into the embryo following oral administration of ethylene glycol during placentation in the rat and rabbit.

In order to evaluate the role of the placenta in the etiology of ethylene glycol (EG) developmental toxicity, the distribution of EG and its main metabolites, glycolic acid (GA) and oxalic acid (OX), into the conceptus was determined at the beginning and completion of placentation in the rat and rabbit. Two groups (n = 28) of timed-pregnant Wistar rats were administered EG (1000 mg/kg bw/day, oral gavage) from gestation day (GD) 6 to either GD 11 or GD 16; similarly, two groups (n = 28) of timed-pregnant New Zealand White rabbits were administered EG from GD 6 to either GD 10 or GD 19. Four animals from each group were sacrificed at 1, 3, 6, 9, 12, 18, or 24 h after the final administration, and maternal blood, extraembryonic fluid, and embryonic tissue were removed for analysis of EG, GA, and OX. The three analytes were predominantly cleared from all compartments in both species within 24 h. Neither EG nor OX preferentially accumulated into the conceptus compartments, compared with the maternal blood, in either species. Critically, GA was preferentially accumulated from the maternal blood only into the rat embryo at GD 11, but not at GD 16 and not into the rabbit embryo at either GD 10 or GD 19. The accumulation of GA into the rat embryo, and its decline over the course of placentation, is discussed in relation to the expression of monocarboxylate transporter isoforms across the syncytiotrophoblast.

Conspiracy beliefs prospectively predict health behavior and well-being during a pandemic.

BACKGROUND: Conspiracy beliefs are associated with detrimental health attitudes during the coronavirus disease 2019 (Covid-19) pandemic. Most prior research on these issues was cross-sectional, however, and restricted to attitudes or behavioral intentions. The current research was designed to examine to what extent conspiracy beliefs predict health behavior and well-being over a longer period of time. METHODS: In this preregistered multi-wave study on a large Dutch research panel (weighted to provide nationally representative population estimates), we examined if conspiracy beliefs early in the pandemic (April 2020) would predict a range of concrete health and well-being outcomes eight months later (December 2020; N = 5745). RESULTS: The results revealed that Covid-19 conspiracy beliefs prospectively predicted a decreased likelihood of getting tested for corona; if tested, an increased likelihood of the test coming out positive; and, an increased likelihood of having violated corona regulations, deteriorated economic outcomes (job loss; reduced income), experiences of social rejection, and decreased overall well-being. Most of these effects generalized to a broader susceptibility to conspiracy theories (i.e. conspiracy mentality). CONCLUSIONS: These findings suggest that conspiracy beliefs are associated with a myriad of negative life outcomes in the long run. Conspiracy beliefs predict how well people have coped with the pandemic over a period of eight months, as reflected in their health behavior, and their economic and social well-being.

A longitudinal analysis of conspiracy beliefs and Covid-19 health responses.

BACKGROUND: Little is known about how conspiracy beliefs and health responses are interrelated over time during the course of the coronavirus disease 2019 (Covid-19) pandemic. This longitudinal study tested two contrasting, but not mutually exclusive, hypotheses through cross-lagged modeling. First, based on the consequential nature of conspiracy beliefs, we hypothesize that conspiracy beliefs predict an increase in detrimental health responses over time. Second, as people may rationalize their behavior through conspiracy beliefs, we hypothesize that detrimental health responses predict increased conspiracy beliefs over time. METHODS: We measured conspiracy beliefs and several health-related responses (i.e. physical distancing, support for lockdown policy, and the perception of the coronavirus as dangerous) at three phases of the pandemic in the Netherlands (N = 4913): During the first lockdown (Wave 1: April 2020), after the first lockdown (Wave 2: June 2020), and during the second lockdown (Wave 3: December 2020). RESULTS: For physical distancing and perceived danger, the overall cross-lagged effects supported both hypotheses, although the standardized effects were larger for the effects of conspiracy beliefs on these health responses than vice versa. The within-person change results only supported an effect of conspiracy beliefs on these health responses, depending on the phase of the pandemic. Furthermore, an overall cross-lagged effect of conspiracy beliefs on reduced support for lockdown policy emerged from Wave 2 to 3. CONCLUSIONS: The results provide stronger support for the hypothesis that conspiracy beliefs predict health responses over time than for the hypothesis that health responses predict conspiracy beliefs over time.

Ideological Responses to the EU Refugee Crisis: The Left, the Right, and the Extremes.

The 2016 European Union (EU) refugee crisis exposed a fundamental distinction in political attitudes between the political left and right. Previous findings suggest, however, that besides political orientation, ideological strength (i.e., political extremism) is also relevant to understand such distinctive attitudes. Our study reveals that the political right is more anxious, and the political left experiences more self-efficacy, about the refugee crisis. At the same time, the political extremes-at both sides of the spectrum-are more likely than moderates to believe that the solution to this societal problem is simple. Furthermore, both extremes experience more judgmental certainty about their domain-specific knowledge of the refugee crisis, independent of their actual knowledge. Finally, belief in simple solutions mediated the relationship between ideology and judgmental certainty, but only among political extremists. We conclude that both ideological orientation and strength matter to understand citizens' reactions to the refugee crisis.

Fear among the extremes: how political ideology predicts negative emotions and outgroup derogation.

The "rigidity of the right" hypothesis predicts that particularly the political right experiences fear and derogates outgroups. We propose that above and beyond that, the political extremes (at both sides of the spectrum) are more likely to display these responses than political moderates. Results of a large-scale sample reveal the predicted quadratic term on socio-economic fear. Moreover, although the political right is more likely to derogate the specific category of immigrants, we find a quadratic effect on derogation of a broad range of societal categories. Both extremes also experience stronger negative emotions about politics than politically moderate respondents. Finally, the quadratic effects on derogation of societal groups and negative political emotions were mediated by socio-economic fear, particularly among left- and right-wing extremists. It is concluded that negative emotions and outgroup derogation flourish among the extremes.

Evaluation of an alternative in vitro test battery for detecting reproductive toxicants in a grouping context.

Previously we showed a battery consisting of CALUX transcriptional activation assays, the ReProGlo assay, and the embryonic stem cell test, and zebrafish embryotoxicity assay as 'apical' tests to correctly predict developmental toxicity for 11 out of 12 compounds, and to explain the one false negative [7]. Here we report on applying this battery within the context of grouping and read across, put forward as a potential tool to fill data gaps and avoid animal testing, to distinguish in vivo non- or weak developmental toxicants from potent developmental toxicants within groups of structural analogs. The battery correctly distinguished 2-methylhexanoic acid, monomethyl phthalate, and monobutyltin trichloride as non- or weak developmental toxicants from structurally related developmental toxicants valproic acid, mono-ethylhexyl phthalate, and tributyltin chloride, respectively, and, therefore, holds promise as a biological verification model in grouping and read across approaches. The relevance of toxicokinetic information is indicated.

A high throughput screening system for predicting chemically-induced reproductive organ deformities.

There is a great need for alternative testing methods for reproductive toxicants that are practical, fast, cost-effective and easy to interpret. Previously we followed a pragmatic approach using readily available tests, which was successful in predicting reproductive toxicity of chemicals [13]. This initial battery still contained apical tests and is fairly complex and low in its throughput. The current study aimed to simplify this screening battery using a mechanistic approach and a panel of high throughput CALUX reporter gene assays. A mechanistic approach was taken to validate this high throughput test battery. To this end it was challenged with two preselected sets of chemicals addressing two major apical effect classes relevant in reproductive toxicity. We found selectivity in this battery in that 82% of the compounds inducing reproductive organ deformities were predicted correctly, while for compounds inducing neural tube defects this was the case in 47% only. This is consistent with the mechanisms of toxicity covered in the battery. The most informative assays in the battery were ERalpha CALUX to measure estrogenicity and the AR-anti CALUX assay to measure androgen receptor antagonism.

Delay and impairment in brain development and function in rat offspring after maternal exposure to methylmercury.

Maternal exposure to the neurotoxin methylmercury (MeHg) has been shown to have adverse effects on neural development of the offspring in man. Little is known about the underlying mechanisms by which MeHg affects the developing brain. To explore the neurodevelopmental defects and the underlying mechanism associated with MeHg exposure, the cerebellum and cerebrum of Wistar rat pups were analyzed by [(18)F]FDG PET functional imaging, field potential analysis, and microarray gene expression profiling. Female rat pups were exposed to MeHg via maternal diet during intrauterinal and lactational period (from gestational day 6 to postnatal day (PND)10), and their brain tissues were sampled for the analysis at weaning (PND18-21) and adulthood (PND61-70). The [(18)F]FDG PET imaging and field potential analysis suggested a delay in brain activity and impaired neural function by MeHg. Genome-wide transcriptome analysis substantiated these findings by showing (1) a delay in the onset of gene expression related to neural development, and (2) alterations in pathways related to both structural and functional aspects of nervous system development. The latter included changes in gene expression of developmental regulators, developmental phase-associated genes, small GTPase signaling molecules, and representatives of all processes required for synaptic transmission. These findings were observed at dose levels at which only marginal changes in conventional developmental toxicity endpoints were detected. Therefore, the approaches applied in this study are promising in terms of yielding increased sensitivity compared with classical developmental toxicity tests.

Developmental immunotoxicity of ethanol in an extended one-generation reproductive toxicity study.

The susceptibility of developing immune system to chemical disruption warrants the assessment of immune parameters in reproductive and developmental testing protocols. In this study, a wide range of immune endpoints was included in an extended one-generation reproduction toxicity study (EOGRTS) design to determine the relative sensitivity of immune and developmental parameters to ethanol (EtOH), a well-known developmental toxicant with immunomodulatory properties. Adult Wistar rats were exposed to EtOH via drinking water (0, 1.5, 4, 6.5, 9, 11.5 and 14 % (w/v EtOH)) during premating, mating, gestation and lactation and continuation of exposure of the F(1) from weaning until killed. Immune assessments were performed at postnatal days (PNDs) 21, 42 and 70. Keyhole limpet hemocyanin (KLH)-specific immune responses were evaluated following subcutaneous immunizations on PNDs 21 and 35. EtOH exposure affected innate as well as adaptive immune responses. The most sensitive immune parameters included white blood cell subpopulations, ConA-stimulated splenocyte proliferation, LPS-induced NO and TNF-α production by adherent splenocytes and KLH-specific immune responses. Most parameters showed recovery after cessation of EtOH exposure after weaning in the 14 % exposure group. However, effects on LPS-induced NO and TNF-α production by adherent splenocytes and KLH-specific parameters persisted until PND 70. The results demonstrate the relative sensitivity to EtOH of especially functional immune parameters and confirm the added value of immune parameters in the EOGRTS. Furthermore, this study identified an expanded KLH-specific parameter set and LPS-induced NO and TNF-α production by adherent splenocytes as valuable parameters that can provide additional information on functional immune effects.

Nasal passages of Göttingen minipigs from the neonatal period to young adult.

Histopathological examination of the nasal passages requires a standardized approach for recording lesion distribution patterns. Nasal diagrams provide guidance to map the lesions. Information on lesions exists for rodents, dogs, and monkeys, which all have been used in inhalation studies. Recently, minipigs have garnered interest as an inhalation model because minipigs resemble humans in many features of anatomy, physiology, and biochemistry and may be a good alternative to monkeys and dogs. The present work explored the microanatomy and histology of the nasal passages of Göttingen minipigs from postnatal day 1 until 6 months of age. Six nasal levels were selected, which allow examination of the squamous, transitional (nonciliated) and ciliated respiratory, and olfactory epithelia; the nasopharynx; and relevant structures such as the vomeronasal organ, olfactory bulb, and nasal/nasopharynx-associated lymphoid tissue.

A baculovirus photolyase with DNA repair activity and circadian clock regulatory function.

Cryptochromes and photolyases belong to the same family of flavoproteins but, despite being structurally conserved, display distinct functions. Photolyases use visible light to repair ultraviolet-induced DNA damage. Cryptochromes, however, function as blue-light receptors, circadian photoreceptors, or repressors of the CLOCK/BMAL1 heterodimer, the transcription activator controlling the molecular circadian clock. Here, we present evidence that the functional divergence between cryptochromes and photolyases is not so univocal. Chrysodeixis chalcites nucleopolyhedrovirus possesses 2 photolyase-like genes: phr1 and phr2. We show that PHR1 and PHR2 are able to bind the CLOCK protein. Only for PHR2, however, the physical interaction with CLOCK represses CLOCK/BMAL1-driven transcription. This result shows that binding of photolyase per se is not sufficient to inhibit the CLOCK/BMAL1 heterodimer. PHR2, furthermore, affects the oscillation of immortalized mouse embryonic fibroblasts, suggesting that PHR2 can regulate the molecular circadian clock. These findings are relevant for further understanding the evolution of cryptochromes and photolyases as well as behavioral changes induced in insects by baculoviruses.

Developmental immunotoxicity in male rats after juvenile exposure to di-n-octyltin dichloride (DOTC).

To determine relevant endpoints for evaluating developmental immunotoxicity due to juvenile exposure and optimal age of the animals at assessment, a wide range of immunological parameters were assessed in a juvenile toxicity study. Rats were exposed to di-n-octyltin dichloride (DOTC) by gavage from postnatal day (PND) 10 through PND 21 and via the diet after weaning using a benchmark dose (BMD) approach. Immune assessments were performed in male rats on PNDs 21, 42, and 70 and a subset of animals was used to evaluate the T-cell dependent antibody response (TDAR) to Keyhole limpet hemocyanin. Immune effects were more pronounced on PND 21 and 42 and observed at lower doses than developmental effects. The most sensitive immune parameters affected included TDAR parameters and thymocyte subpopulations with lower confidence limits of the benchmark doses (BMDLs) below the overall no-observed-adverse-effect-level (NOAEL) for DOTC reported so far in literature. These findings illustrate the relative sensitivity of the developing immune system for DOTC, the additional value of assessing functional immune parameters, and underscore the relevance of juvenile immunotoxicity testing in view of the risk assessment of chemicals.

The Potorous CPD photolyase rescues a cryptochrome-deficient mammalian circadian clock.

Despite the sequence and structural conservation between cryptochromes and photolyases, members of the cryptochrome/photolyase (flavo)protein family, their functions are divergent. Whereas photolyases are DNA repair enzymes that use visible light to lesion-specifically remove UV-induced DNA damage, cryptochromes act as photoreceptors and circadian clock proteins. To address the functional diversity of cryptochromes and photolyases, we investigated the effect of ectopically expressed Arabidopsis thaliana (6-4)PP photolyase and Potorous tridactylus CPD-photolyase (close and distant relatives of mammalian cryptochromes, respectively), on the performance of the mammalian cryptochromes in the mammalian circadian clock. Using photolyase transgenic mice, we show that Potorous CPD-photolyase affects the clock by shortening the period of behavioral rhythms. Furthermore, constitutively expressed CPD-photolyase is shown to reduce the amplitude of circadian oscillations in cultured cells and to inhibit CLOCK/BMAL1 driven transcription by interacting with CLOCK. Importantly, we show that Potorous CPD-photolyase can restore the molecular oscillator in the liver of (clock-deficient) Cry1/Cry2 double knockout mice. These data demonstrate that a photolyase can act as a true cryptochrome. These findings shed new light on the importance of the core structure of mammalian cryptochromes in relation to its function in the circadian clock and contribute to our further understanding of the evolution of the cryptochrome/photolyase protein family.

Normal anatomy and histology of the adult zebrafish.

The zebrafish has been shown to be an excellent vertebrate model for studying the roles of specific genes and signaling pathways. The sequencing of its genome and the relative ease with which gene modifications can be performed have led to the creation of numerous human disease models that can be used for testing the potential and the toxicity of new pharmaceutical compounds. Many pharmaceutical companies already use the zebrafish for prescreening purposes. So far, the focus has been on ecotoxicity and the effects on embryonic development, but there is a trend to expand the use of the zebrafish with acute, subchronic, and chronic toxicity studies that are currently still carried out with the more conventional test animals such as rodents. However, before we can fully realize the potential of the zebrafish as an animal model for understanding human development, disease, and toxicology, we must first greatly advance our knowledge of normal zebrafish physiology, anatomy, and histology. To further this knowledge, we describe, in the present article, location and histology of the major zebrafish organ systems with a brief description of their function.

Kinetics of cyclobutane thymine dimer splitting by DNA photolyase directly monitored in the UV.

CPD photolyase uses light to repair cyclobutane pyrimidine dimers (CPDs) formed between adjacent pyrimidines in UV-irradiated DNA. The enzyme harbors an FAD cofactor in fully reduced state (FADH(-)). The CPD repair mechanism involves electron transfer from photoexcited FADH(-) to the CPD, splitting of its intradimer bonds, and electron return to restore catalytically active FADH(-). The two electron transfer processes occur on time scales of 10(-10) and 10(-9) s, respectively. Until now, CPD splitting itself has only been poorly characterized by experiments. Using a previously unreported transient absorption setup, we succeeded in monitoring cyclobutane thymine dimer repair in the main UV absorption band of intact thymine at 266 nm. Flavin transitions that overlay DNA-based absorption changes at 266 nm were monitored independently in the visible and subtracted to obtain the true repair kinetics. Restoration of intact thymine showed a short lag and a biexponential rise with time constants of 0.2 and 1.5 ns. We assign these two time constants to splitting of the intradimer bonds (creating one intact thymine and one thymine anion radical T(∘-)) and electron return from T(∘-) to the FAD cofactor with recovery of the second thymine, respectively. Previous model studies and computer simulations yielded various CPD splitting times between < 1 ps and < 100 ns. Our experimental results should serve as a benchmark for future efforts to model enzymatic photorepair. The technique and methods developed here may be applied to monitor other photoreactions involving DNA.

Developmental immunotoxicity of di-n-octyltin dichloride (DOTC) in an extended one-generation reproductive toxicity study.

Developmental immunotoxicity assessment is considered ready for inclusion in developmental toxicity studies. Further evaluation of proposed and additional assays is needed to determine their utility in assessing developmental immunotoxicity. In this study, a wide range of immunological parameters was included in an extended one-generation reproductive toxicity protocol. F(0) Wistar rats were exposed to DOTC via the feed (0, 3, 10, and 30mg/kg) during pre-mating, mating, gestation and lactation and subsequently F(1) were exposed from weaning until sacrifice. Immune assessments by several immune parameters were performed at PNDs 21, 42 and 70. The T cell-dependent antibody response to Keyhole Limpet hemocyanin (KLH) was assessed following subcutaneous immunizations with KLH on PNDs 21 and 35 and the delayed-type hypersensitivity response (DTH) against KLH was evaluated at PND 49. No effects were found on PND 21. While effects on lymphocyte subpopulations in the thymus were only observed in the 30mg/kg group on PND 42, effects on lymphocyte subpopulations in the spleen were found in the 30mg/kg group on both PNDs 42 and 70. The DTH response already showed an effect at 3mg/kg and was the overall critical endpoint. The results from this study support the inclusion of splenocyte subpopulation parameters in developmental toxicity studies and identified the DTH response as an important functional parameter.

Developmental immunotoxicity of methylmercury: the relative sensitivity of developmental and immune parameters.

Current developmental and reproductive toxicity protocols include only a limited set of parameters for effects on the developing immune system. In this study, a wide range of immunological parameters were included in a pre- and postnatal developmental toxicity study. Dose-response data were compared to determine the relative sensitivity of different immune and developmental parameters. Mated female Wistar rats were dosed daily by gavage with methylmercury (0, 0.1, 0.4, 0.7, 1.0, 1.5, and 2.0 mg/kg BW/day) from gestational day 6 to postnatal day (PND) 10. In addition to general, reproductive, and developmental parameters, a wide range of immunological parameters were assessed in male offspring at PNDs 21, 42, and 70. The T cell-dependent antibody response to keyhole limpet hemocyanin (KLH) was assessed following sc immunizations on PNDs 21 and 35. Dose-response data were analyzed using the benchmark dose (BMD) approach by fitting dose-response models to the various endpoints. Methylmercury induced effects on developmental parameters, such as growth parameters and pup mortality. Effects on the immune system were found at doses without observed developmental toxicity. Immune effects differed at the three time points and consisted mainly of effects on functional parameters. The parameter with the lowest 5% lower confidence bound of the BMD (BMDL) was the primary KLH-specific IgG antibody response, which showed a dose-dependent decrease with a BMD of 0.039 mg/kg BW/day (CI 0.010-0.12). These data show the relatively high sensitivity of the developing immune system and thereby illustrate the relevance of testing immune parameters in reproductive and developmental toxicity testing protocols.

DNA photolyases of Chrysodeixis chalcites nucleopolyhedrovirus are targeted to the nucleus and interact with chromosomes and mitotic spindle structures.

Cyclobutane pyrimidine dimer (CPD) photolyases convert UV-induced CPDs in DNA into monomers using visible light as the energy source. Two phr genes encoding class II CPD photolyases PHR1 and PHR2 have been identified in Chrysodeixis chalcites nucleopolyhedrovirus (ChchNPV). Transient expression assays in insect cells showed that PHR1-EGFP fusion protein was localized in the nucleus. Early after transfection, PHR2-EGFP was distributed over the cytoplasm and nucleus but, over time, it became localized predominantly in the nucleus. Immunofluorescence analysis with anti-PHR2 antiserum showed that, early after transfection, non-fused PHR2 was already present mainly in the nucleus, suggesting that the fusion of PHR2 to EGFP hindered its nuclear import. Both PHR-EGFP fusion proteins strongly colocalized with chromosomes and spindle, aster and midbody structures during host-cell mitosis. When PHR2-EGFP-transfected cells were superinfected with Autographa californica multiple-nucleocapsid NPV (AcMNPV), the protein colocalized with virogenic stroma, the replication factories of baculovirus DNA. The collective data support the supposition that the PHR2 protein plays a role in baculovirus DNA repair.

DNA repair by photolyase: a novel substrate with low background absorption around 265 nm for transient absorption studies in the UV.

CPD photolyase enzymatically repairs the major UV-induced lesion in DNA, the cyclobutane pyrimidine dimer (CPD), by photoreversion of the damage reaction. An enzyme-bound reduced flavin (FADH(-)) cofactor functions as photosensitizer. Upon excitation, it transiently transfers an electron to the CPD, triggering scission of the interpyrimidine bonds. After repair completion, the electron returns to the flavin to restore its functional reduced form. A major difficulty for time-resolved spectroscopic monitoring of the enzymatic repair reaction is that absorption changes around 265 nm accompanying pyrimidine restoration are obscured by the strong background absorption of the nondimerized bases in DNA. Here we present a novel substrate for CPD photolyase that absorbs only weakly around 265 nm: a modified thymidine 10-mer with a central CPD and all bases, except the one at the 3' end, replaced by 5,6-dihydrothymine which virtually does not absorb around 265 nm. Repair of this substrate by photolyases from Anacystis nidulans and from Escherichia coli was compared with repair of two conventional substrates: a 10-mer of unmodified thymidines containing a central CPD and an acetone-sensitized thymidine 18-mer that contained in average six randomly distributed CPDs per strand. In all cases, the novel substrate was repaired with an efficiency very similar to that of the conventional substrates (quantum yields in the order of 0.5 upon excitation of FADH(-)). Flash-induced transient absorption changes at 267 nm could be recorded on a millisecond time scale with a single subsaturating flash and yielded very similar signals for all three substrates. Because of its low background absorption around 265 nm and the defined structure, the novel substrate is a promising tool for fast and ultrafast transient absorption studies on pyrimidine dimer splitting by CPD photolyase.

Quantum yield measurements of short-lived photoactivation intermediates in DNA photolyase: toward a detailed understanding of the triple tryptophan electron transfer chain.

The light-dependent DNA repair enzyme photolyase contains a unique evolutionary conserved triple tryptophan electron transfer chain (W382-W359-W306 in photolyase from E. coli) that bridges the approximately 15 A distance between the buried flavin adenine dinucleotide (FAD) cofactor and the surface of the protein. Upon excitation of the semireduced flavin (FADH(o)), electron transfer through the chain leads to formation of fully reduced flavin (FADH(-); required for DNA repair) and oxidation of the most remote tryptophan residue W306, followed by its deprotonation. The thus-formed tryptophanyl radical W306(o)(+) is reduced either by an extrinsic reductant or by reverse electron transfer from FADH(-). Altogether the kinetics of these charge transfer reactions span 10 orders of magnitude, from a few picoseconds to tens of milliseconds. We investigated electron transfer processes in the picosecond-nanosecond time window bridging the time domains covered by ultrafast pump-probe and "classical" continuous probe techniques. Using a recent dedicated setup, we directly show that virtually no absorption change between 300 ps and 10 ns occurs in wild-type photolyase, implying that no charge recombination takes place in this time window. In contrast, W306F mutant photolyase showed a partial absorption recovery with a time constant of 0.85 ns. In wild-type photolyase, the quantum yield of FADH(-) W306(o)(+) was found at 19 +/- 4%, in reference to the established quantum yield of the long-lived excited state of [Ru(bpy)(3)](2+). With this yield, the optical spectrum of the excited state of FADH(o) can be constructed from ultrafast spectroscopic data; this spectrum is dominated by excited state absorption extending from below 450 to 850 nm. The new experimental results, taken together with previous data, allow us to propose a detailed kinetic and energetic scheme of the electron transfer chain.