Photochemistry and Photobiology, 2009, 85: 78–87
Crayfish Procambarus clarkii Retina and Nervous System Exhibit
Antioxidant Circadian Rhythms Coupled with Metabolic and
Luminous Daily Cycles
Marı́a Luisa Fanjul-Moles*, Julio Prieto-Sagredo, Dario Santiago López, Ramón
Bartolo-Orozco and Hugo Cruz-Rosas
Laboratorio de Neurofisiologı́a Comparada, Facultad de Ciencias, Universidad Nacional
Autónoma de México, Mexico City, México
Received 10 December 2007, accepted 5 May 2008, DOI: 10.1111 ⁄ j.1751-1097.2008.00399.x
ABSTRACT
Based on previous work in which we proposed midgut as a
putative peripheral oscillator responsible for circadian reduced
glutathione (GSH) crayfish status, herein we investigated the
retina and optic lobe-brain (OL-B) circadian GSH system and
its ability to deal with reactive oxygen species (ROS) produced
as a consequence of metabolic rhythms and light variations. We
characterized daily and antioxidant circadian variations of the
different parameters of the glutathione system, including GSH,
oxidized glutathione (GSSG), glutathione reductase (GR) and
glutathione peroxidase (GPx), as well as metabolic and lipoper-
oxidative circadian oscillations in retina and OL-B, determining
internal and external GSH-system synchrony. The results
demonstrate statistically significant bi- and unimodal daily and
circadian rhythms in all GSH-cycle parameters, substrates and
enzymes in OL-B and retina, as well as an apparent direct effect
of light on these rhythms, especially in the retina. The luminous
condition appears to stimulate the GSH system to antagonize
ROS and lipid peroxidation (LPO) daily and circadian rhythms
occurring in both structures, oscillating with higher LPO under
dark conditions. We suggest that the difference in the effect of
light on GSH rhythmic mechanisms of both structures for
antagonizing ROS could be due to differences in glutathione-
system coupling strength with the circadian clock.
INTRODUCTION
The rotation movement of the Earth originates changes in
environmental conditions over the 24 h period, the most
prominent being the light–darkness (LD) cycle. The predict-
ability of these changes allows organisms to keep track of time
by anticipating these changes and adjusting their internal
temporal order to the external time. In organisms, this ability
underlies the capacity for endogenous temporal organization
of cellular process over the course of the approximately 24 h
period. Cellular machinery that generates this ability is known
as the biologic clock, and its output, as circadian rhythm. The
circadian system in its function as an endogenous clock
*Corresponding author email: mlfm@hp.fciencias.unam.mx (Marı́a Luisa
Fanjul-Moles)
 2008 The Authors. Journal Compilation. The American Society of Photobiology 0031-8655/09
78
entrains the rhythms to seasonal photoperiod changes, max-
imizing metabolic and behavioral functions. In animals,
circadian and exogenous daily variations including those
related to locomotor and brain activity, as well as temperature
and light fluctuations, result in corresponding daily patterns of
reactive oxygen species (ROS) (1), which should lead to
rhythmic oxidative damage in the absence of a rhythmic
antioxidant system to counterbalance this. ROS and anti-
oxidants are known to influence the expression of a number of
genes and transduction pathways (2). It has recently been
demonstrated that the redox process plays a central role in the
function of the master clock of different organisms (3). In
addition, some authors propose that circadian clocks organize
metabolic functions into a coherent daily schedule, assuring
the synchrony of this schedule with environmental changes (4).
The crayfish is a nocturnal organism that exhibits a span of
overt circadian rhythms for adaptation to daily and seasonal
environmental light variation. In Procambarus clarkii, many
authors have documented metabolic (5–7) and activity (8,9)
circadian rhythms that should produce daily variations in the
oxidative status (OS) of this animal’s internal environment. Of
late, daily and circadian changes in OS produced by different
light conditions and its counterbalance by an antioxidant
circadian system—the glutathione system—have been docu-
mented in the midgut and hemolymph of P. clarkii (10,11).
These works revealed differences between the midgut and
hemolymph reduced glutathione (GSH) oscillatory system that
suggest differences in the coupling strength of both organs
with the crayfish circadian clock. These results led us to
propose the hemolymph as a passive system responding to an
oscillating driving force depending on various self-sustaining
oscillators such as the midgut, and those proposed as putative
pacemakers of crayfish and located in the brain, optic lobe and
the retina (12). However, neither the existence of a GSH
circadian system in these putative pacemakers nor the efficacy
of this to counterbalance oxidative damage of internal and
external ROS in these neural structures, as well as its timing,
have been studied. Thus, in this work we investigated the
interaction of metabolic, environmental, and GSH antioxidant
rhythmic changes with changes in oxidative damage in the
aforementioned structures and proposed, as putative pace-
makers of the crayfish circadian system, retina and the
complex optic lobe-brain (OL-B).

MATERIALS AND METHODS
Animals and experimental design. We used 72 P. clarkii crayfish of
homogenous size and weight in intermolt stage and that were
acclimatized to the laboratory for 1 month in aquariums placed under
natural LD cycle conditions at a temperature of 20C, pH 7.9 and O2
concentration of 5.7 mg L)1. The aquariums were provided with
polyvinyl chloride tubes simulating burrows, which allowed the
animals to hide from the light. After acclimatization, the animals
were divided into three groups and sub-divided into two batches each
for two experiments under the following different light-cycle condi-
tions: (1) 36 animals submitted to LD 12 h:12 h of low intensity
(0.043 W m)2) for 15 days, and at the end killed, and (2) 36 animals
treated as described previously and subsequently exposed to dark–dark
(DD) for 72 h. At the end of each condition, six specimens from each
group were selected at random six times daily in a 24 h cycle. At each
experimental time point, each organism was anesthetized by placing it
on ice in the dark and each was subsequently killed. Retina and OL-B
were dissected and processed. The first group was processed to
determine GSH and oxidized glutathione (GSSG), while the second
group was employed to determine glutathione peroxidase (GPx),
glutathione reductase (GR) and lipid peroxidation (LPO). In addition,
a third group was utilized to determine L-glucose.
Tissue sample. For GSH and GSSG determination, animals were
killed by decapitation, and OL-B and retina were dissected and
homogenated separately with a tissue grinder in 400 lL 10% trichlo-
roacetic acid. Samples were centrifuged for 15 min (12 800 g) at 8C.
The supernatant was stored at )70C until GSH and GSSG were
determined. Retina and OL-B samples for enzyme, LPO and glucose
determinations were homogenized in 500 lL phosphate buffer (pH 8),
centrifuged as previously described, and stored at )70C until assayed.
For hemolymph glucose determination, 25 lL samples were obtained
with a micropipette from a small hole punched into the ventral
membrane of the second or third abdominal segment, 100 lL
phosphate buffer (pH 8) was added, and the sample was centrifuged
and stored as described previously.
GSH and GSSG high-performance liquid chromatography (HPLC)
determination. Reduced glutathione and GSSG were determined
simultaneously with the reverse-phase method previously reported by
Harvey et al. (13) and modified at our laboratory for amperometric
methods. All reagents were obtained from Sigma-Aldrich Co.
(St. Louis, MO). GSH and GSSG standards were employed to
construct calibration curves, and L-cysteine was used as internal
standard. Stock solutions of these reagents were prepared each week,
and dilutions for calibration curves were prepared daily. The mobile
phase consisted of a sodium phosphate (monobasic) 10 lM solution
adjusted to 2.7 pH with 85% phosphoric acid to which 0.05 mM octyl
sulfate and 2% acetonitrile were added. The mobile phase was filtered
with 0.22 lm nylon membrane and degassed in line with the LC26B
model On-line Vacuum Degasser, BAS (all Bioanalytical Systems
[BAS] equipment, Bioanalytical Systems, Inc., West Lafayette, IN). An
isocratic pump model PM 80 Solvent Delivery System BAS was
utilized at a 0.5 mL min)1 rate to deliver the eluent. A 100 lL volume
of both standards and samples was injected using a Rheodyne I125
injector (20 lL loop, Rheodyne LLC, Rohnert Park, CA), and
separation was performed with a C18 BAS column model MF-8954
(3 · 10 mm, 3 lm, ODS 80Å). Detection was performed with a LC4C
model Amperometric Detector BAS using a glassy carbon dual
electrode and an Ag-AgCl reference electrode. Detection parameters
were as follows: applied voltage, 1.35 V; generator voltage, 0.95 V;
detection range, 0.5–2 lA and filter, 0.1 Hz. Detected currents were
recorded in a BAS Dual Pen Recorder model 1202-9054. GSH and
GSSH peak amplitudes were measured, and the concentration was
determined interpolating data in the calibration curves; the
GSH ⁄ GSSG ratio was calculated.
GR and GPx activity and protein assay. GR activity was determined
as previously reported (14) employing the method described by
Bompart et al. (15). Briefly, 20 lL of the total 500 lL homogenate
was placed in a cuvette containing GSSG 0.44 mmol L)1 in 0.1 M
phosphate buffer 0.03 M EDTA at pH 7 and reduced 0.036 M
nicotinamide adenine dinucleotide phosphate (NADPH) prepared
and added immediately prior to the assay as the starting reagent. The
assay was run at 340 nm for 4 min with absorbance readings taken
every 30 s. Absorbance was assayed for all readings in an Ultraspec
Photochemistry and Photobiology, 2009, 85 79
2000 spectrophotometer (Pharmacia Biotech, Buckinghamshire, UK).
Activity was expressed in millimoles of oxidized NADPH per g of
protein per min using an extinction coefficient of 6.2 mM min)1. GPx
activity was determined using the method described by Paglia and
Valentine (16). Twenty microliters of the homogenate was placed in a
cuvette containing 0.001 M sodium azide (NaN3) in 0.1 M phosphate
buffer 0.001 M EDTA at pH 7.4; GSH 0.001 M, NADPH 0.2 mM and
GR 1 U mL)1 were then prepared and added immediately prior to the
assay; 30 lL of 0.25 mM perhydrol was also prepared and added
immediately prior to assay initiation as the starting reagent. The assay
was run at 340 nm for 10 min, with absorbance readings taken every
1 min. Absorbance was assayed for all readings in an Ultraspec 2000
spectrophotometer (Pharmacia Biotech). Activity was expressed in
millimoles of oxidized NADPH per g of protein per min utilizing an
extinction coefficient of 6.2 mM min)1. The protein assay was per-
formed according to Bradford.
Glucose determination. Hemolymph (50 lL) as well as retina and
eyestalk tissue were collected from each animal at the base of a walking
leg every 4 h over a 24 h period. Glucose levels were measured by
Trinder’s glucose oxidase method (17) utilizing a Diagnostic Chemical
Limited kit (Charlottetown, CA). Absorbance at 505 nm was assayed
in an Ultraspec 2000 spectrophotometer (Pharmacia Biotech).
Lipid peroxidation analysis. LPO levels were quantified utilizing a
K-ASSAY LPO kit (Kamiya Biomedical Co., Seattle, WA) (18). Six
animals from each group were randomly chosen and killed by
decapitation. Retina and OL-B samples were homogenized in PBS
1:10 and stored at )70C until the day of the assay to measure LPO.
The homogenates of each sample were centrifuged. The supernatant
was removed and the enzyme reagent (ascorbic oxidase and lipoprotein
lipase) was added. The mixture was incubated for 5 min at a
temperature of 30C and the chromogen reagent (10-N-methylcarba-
moyl-3,7-dimethylamino-10-H-phenothiazine) was added. The result-
ing mixture was incubated for 10 min at 30C. Finally, absorbance was
measured at 675 nm in a spectrophotometer. A two-point calibration
curve was done using the saline blank (0 nmol mL)1) and the
50 nmol mL)1 cumene hydroperoxidase standard provided with the
kit. Results were then calculated using the kit instructions.
Assessment of light parameters. The photoperiod was provided by
neon lamps turned on and off by a timer at 0700 and 1900 h, and light
quantum scalar irradiance was calibrated with a photoradiometer
equipped with a spherical submarine sensor (LiCor models LI-189 and
LI-193SA; LiCor, Lincoln, NE). Intensity values were set at the
minimal values of light irradiance to which crayfish are exposed in
their natural environment.
Data analysis. Chronograms were constructed as group
mean ± standard deviation (SD). To estimate circadian rhythms for
each GSH parameter (GSH, GSSG, GSH ⁄ GSSG, GR and GPx) of the
different groups of animals, a single cosinor analysis was performed.
The COSANA software program was used. Based on a test period (s),
cosinor analysis adjusts data to a cosinusoidal function and provides
an objective test of whether the rhythm amplitude differs from zero,
i.e. whether the rhythm is validated for an assumed s (19). This method
provides descriptive estimators for a number of different rhythm
parameters, i.e. acrophase, mesor, amplitude and percentage of
rhythmicity (PR). Acrophase is the crest time of the best-fitting
mathematical function approximating the data, while mesor comprises
the value around which oscillation occurs; when the time interval
between data samplings is constant, it equals the rhythmic oscillation’s
arithmetic mean. Hence, in the present work it corresponds to the
arithmetic mean of the rhythmic oscillation of the concentration or
activity of the GSH parameters over a 24 h period. Mesor (M) is
defined as the adjusted mean of the rhythm. When the interval of time
between data sampling (Dt) is constant, M equals the arithmetic mean.
The amplitude is equal to one half of the difference between highest
and lowest oscillation values, and PR is the percentage of data
included within the 95% confidence limits of the best-fitting cosine
function. This test allows subjective examination of the hypothesis that
rhythm amplitude differs from zero using different trial-period lengths.
In the current work, several periods were tested to analyze whether
temporal GSH-parameter profiles under different LD conditions were
indeed circadian. In addition, analysis of variance (ANOVA) was
followed by Scheffé post hoc comparisons for rhythms for which
waveform did not adjust cosinor. For statistical analysis of differences
in terms of the concentration and activity of all parameters between
80 Marı́a Luisa Fanjul-Moles et al.
LD and DD in retina and OL-B, a Student’s t-test was performed.
Significant clustering of phases in both LD and DD were determined
using circular statistics (Rayleigh and V-tests). Both tests examine
whether there is statistical evidence of directional phase preference in
the population from which the sample is drawn (20).
RESULTS
Analysis of crayfish retina and OL-B complex extracts revealed
the presence of two substances that oxidized at 1.35 V and
eluted at the same time as the GSH and GSSG standard (mean
elution time = 4 and 8 min, respectively). In addition, a single
symmetrical and additive peak was observed for both sub-
stances when GSH and GSSG external standards were added
to acid extracts in each injection (Fig. 1). The voltage–
amperage curve generated for GSH and GSSG standards
and presumptive peaks in the sample were also identical. Based
on these criteria, these two peaks were identified as GSH and
GSSG.
Results of the current study demonstrate the presence of all
GSH-system components in crayfish retina and OL-B. These
structures show lower GSH and GSSG concentration values
than those reported for hemolymph and midgut in the same
species (10) as follows: LD retina GSH = 42.6 ± 3.7 lM and
GSSG = 8.6 ± 1.0 lM; DD retina GSH = 14.96 ± 1 lM
and GSSG = 6.12 ± 0.8 lM; LD OL-B GSH = 26.4 ±
2.0 lM and GSSG = 2.35 ± 0.2 lM; and DD OL-B GSH =
12.5 ± 0.8 lM and GSSG = 7.3 ± 1.2 lM. Glutathione
Figure 1. (a) Chromatograms from 150 and 300 ng external standards of GSH and GSSH. Retention times are 4 and 8 min, respectively. (b)
HPLC of an acid extract of crayfish eyestalk-brain (OL-B). (c) Separation of the same acid extract with 100 and 200 ng GSH and GSSG external
standards. Inset shows the mobile phase and TCA chromatograms. Vertical arrows indicate the injection time, diagonal arrows show GSH, GSSG
and TCA peaks. For details, see Materials and Methods.
reductase and GPx also demonstrated lower activity than that
reported in the same work for hepatic tissue as follows: LD
retina GR = 11.7 ± 2.5 lM NADPH ox g)1 prot min)1;
GPX = 2.4 ± 0.4 lM NADPH ox g)1 prot min)1; DD retina
GR = 17.1 ± 4.2 lM NADPH ox g)1 prot min)1; GPx =
10.2 ± 1 lM NADPH ox g)1 prot min)1; LD OL-B
GR = 37.4 ± 4.9 lM NADPH ox g)1 prot min)1; GPx =
8.3 ± 1.9 lM NADPH ox g)1 prot min)1; DD OL-B,
GR = 6.7 ± 1.3 lM NADPH ox g)1 prot min)1; and GPx =
15.33 ± 3.8 lM NADPH ox g)1 prot min)1. The amount
and activity of all glutathione system parameters changed with
the luminous condition. The Student’s t-test revealed signifi-
cant differences in all parameters except GSSG and GR
between LD and DD in brain and retina, respectively (Table 1).
Daily and circadian rhythm determination
Chronograms showing GR and GPx temporal activity in
OL-B are depicted in Fig. 2a,b. In LD, the GR activity peak is
at 0800 h, apparently increasing immediately after lights on at
0700 h, with a second peak appearing at 2000 h after lights off
at 1900 h. Cosinor analysis revealed a GR bimodal significant
daily rhythm with a 12 h period value (Table 2). Both peaks
are coincident with maximal peaks of GPx bimodal activity,
although GPx showed lower average activity throughout 24 h
(M = 8.6) than GR (M = 36.9 ± 3.8) (Table 2). After 72 h
of darkness, the mean GPx activity level increases (M = 16.1),
 Photochemistry and Photobiology, 2009, 85 81

exhibiting a bimodal rhythm of s = 12.3 h, which peaks at In the retina, changes in GR and GPx activity in LD and
1200 h. Meanwhile, GR-activity dampens showing no statis- DD were observed (Fig. 2c,d). GR activity oscillates under
tically significant oscillation (M = 6.7, A = 3.14). LD, exhibiting a bimodal daily rhythm not statistically
significant neither by cosinor or ANOVA (Table 2). GPx
Table 1. Mean and standard error for the variables in LD and DD activity depicts a statistically significant bimodal rhythm
conditions. (s = 12.3 h) that establishes a 4 h phase relationship (w) with
GR rhythmic oscillation. Maximal GR peak activity takes
Variable LD 12:12 Constant darkness
place at photophase at the middle of the subjective day, after
OL-B the second lower peak of GPx activity at 0800 h after lights on.
GSH1* 26.42 ± 2.04 12.57 ± 0.85 However, under darkness, a GR statistically significant circa-
GSSG* 2.35 ± 0.2 7.33 ± 1.27 dian rhythm emerges (t = 24.3); meanwhile GPx rhythmic
GSH ⁄ GSSG* 13.35 ± 1.35 2.83 ± 0.42
activity seems to disappear (Table 2).
GR2* 37.4 ± 4.9 6.7 ± 1.3
GPx* 8.3 ± 1.9 15.33 ± 3.8 Temporal GSH and GSSG concentration changes in
LPO3* 5.76 ± 0.62 26.82 ± 0.96 OL-B complex are shown in Fig. 3a,b. Figure 3a depicts
Glucose4 1 ± 0.12 1.13 ± 0.18 chronograms showing 24 h LD variations of GSH, GSSG
Total protein5* 245 ± 16.5 801.1 ± 15.4 and GSH ⁄ GSSG ratio; cosinor analysis detected no 24 h
Retina
GSH* 42.67 ± 3.75 14.96 ± 1.08 circadian significant rhythms either in GSH or in GSSG
GSSG 8.64 ± 1.14 6.12 ± 0.82 abundance, but did detect bimodal oscillations with period
GSH ⁄ GSSG* 6.2 ± 0.58 3.31 ± 0.31 values of s = 12.5 and 11.9 h, respectively. The corresponding
GR* 11.7 ± 2.5 17.1 ± 4.2 chronograms show GSH and GSSG damped bimodal oscilla-
GPx* 2.47 ± 0.38 10.2 ± 1.08
tions, which shows an increasing GSH ⁄ GSSG ratio in mid-
LPO* 31.51 ± 1.32 509.87 ± 23.31
Glucose 0.52 ± 0.17 0.44 ± 0.06 photophase at the lowest GSSG oscillation value, followed by
Total protein* 230 ± 20 658.3 ± 6.5 a second peak at 0400 h after GSH maximal peak at the
Hemolymph subjective night. After 72 h in DD (Fig. 3c), GSH rhythm
Glucose 19.61 ± 2.24 20.43 ± 3.29 amplitude decreases, but this tripeptide abundance exhibits
statistically significant bimodal rhythm with a 12 h period
Units of measure: 1GSH and GSSG, lM; 2GR and GPx, lM NADPH value (Table 2) with maximal peak at 2000 h and a second
ox g)1 prot min)1; 3LPO, nmol mL)1; 4glucose, mM; 5protein, lg per
structure. *Student’s t-test revealed statistically significant differences at 0800 h. In contrast, GSSG oscillation amplitude under
between LD and DD means for each variable (P < 0.01). this dark condition increases, indicating an increment of

Figure 2. Chronograms showing rhythmic enzymatic activity of optic lobe-brain (a, b) and retina (c, d) under 12:12 light–dark cycles and
continuous darkness. All data are mean ± standard error (SE) (n = 6). Upper black and white bars in each graph denote light and dark phase.
82 Marı́a Luisa Fanjul-Moles et al.

Table 2. Cosinor analysis for the brain and retina variables.

Variable ⁄ parameter Period (h) Mesor1 Amplitude1 Acrophase2 (h) PR (%) P

OL-B LD
GSH 12.5* 26.97 ± 1.72 9.1 ± 2.45 02:52 ± 0:32 34.7 0.01
GSSG 11.9* 2.34 ± 0.17 0.81 ± 0.24 05:35 ± 0:35 30.26 0.01
GSH ⁄ GSSG 243 13.31 1.87 11:29 3.46 0.63
GR 12* 36.9 ± 3.8 24.26 ± 5.4 07:40 ± 0:26 43.7 0.01
GPx 12* 8.6 ± 1.7 6.13 ± 2.5 07:21 ± 0:46 18.9 0.05
LPO 244 5.37 1.49 05:43 9.5 0.26
Glucose 12.8* 1.01 ± 0.11 0.46 ± 0.15 02:30 ± 0:41 25 0.05
OL-B DD
GSH 12* 12.57 ± 0.57 3.39 ± 1.06 08:26 ± 0:36 27.37 0.01
GSSG 26.85 7.72 3.76 23:51 14.73 0.12
GSH ⁄ GSSG 11.46 2.82 1.07 03:34 11.75 0.22
GR 127 6.7 3.14 08:45 9.2 0.2
GPx 12.3* 16.14 ± 3.43 14.49 ± 4.82 10:16 ± 0:39 29.2 0.02
LPO 24.4* 26.81 ± 0.85 3.74 ± 1.19 22:27 ± 1:15 26.83 0.01
Glucose 12.3* 0.98 ± 0.08 0.6 ± 0.11 9:17 ± 0:21 54.19 0.01
Retina LD
GSH 12* 42.67 ± 2.35 22.74 ± 3.32 12:19 ± 0:17 63.44 0.01
GSSG 24* 8.65 ± 0.9 5.7 ± 1.27 10:23 ± 0:51 42.74 0.01
GSH ⁄ GSSG 24* 6.2 ± 0.39 3.36 ± 0.55 00:11 ± 0:38 57.61 0.01
GR 128 11.5 5.8 11:23 16.7 0.2
GPx 12.3* 2.48 ± 0.32 1.58 ± 0.44 08:20 ± 0:35 35.67 0.01
LPO 12.1* 31.71 ± 0.95 7.09 ± 1.33 02:20 ± 0:22 52.11 0.01
Glucose 24* 0.35 ± 0.04 0.2 ± 0.06 23:07 ± 1:06 26.32 0.01
Retina DD
GSH 21.49 14.79 3.21 13:16 14.75 0.1
GSSG 11.9* 6.1 ± 0.66 3.92 ± 0.94 04:25 ± 0:27 39.12 0.01
GSH ⁄ GSSG 12* 3.31 ± 0.27 1.26 ± 0.38 09:18 ± 0:35 28.69 0.01
GR 24.3* 17.3 ± 3.2 17.7 ± 4.5 03:14 ± 1:00 36.3 0.01
GPx 1210 10.3 3.36 4:39 16.62 0.09
LPO 21.4* 505.47 ± 21.9 77.88 ± 31.3 12:43 ± 1:20 18.7 0.05
Glucose 12.6* 0.45 ± 0.05 0.24 ± 0.07 01:17 ± 0:35 35.13 0.01
Hemolymph LD
Glucose 22.8* 1.1 ± 0.11 0.44 ± 0.16 16:03 ± 1:19 21.02 0.05
Hemolymph DD
Glucose 23.5* 1.13 ± 0.17 0.61 ± 0.24 12:38 ± 1:27 19 0.05

PR, percentage of rhythm. P, cosinor statistical significance. 1Units of measure: GSH and GSSG: mM; GR and GPx: lM NADPH g)1 protein min)1;
LPO: nmol mL)1; glucose: mg dL)1. 2External time. 3F = 2.16, P > 0.05. LSD post hoc: 1200 h vs 8, 16, 20, 24 h, P < 0.05. 4F = 2.05, P > 0.05.
LSD post hoc: 800 h vs 12, 16 h, P < 0.05. 5F = 2.1, P > 0.05. LSD post hoc: 400 h vs 12, 16, 24 h, P < 0.05; 2000 vs 16, 24 h, P < 0.05.
6
F = 1.05, P > 0.05. 7F = 0.6, P > 0.05. 8F = 0.7, P > 0.05. 9F = 2.662, P < 0.05. LSD post hoc: 400 h vs 8, 12, 16, 20, 24 h, P < 0.05.
10
F = 1.011, P > 0.05. LSD post hoc: 400 h vs 8, 12, 20 h, P < 0.05. *Statistically significant by cosinor.

ROS. Although ANOVA and Scheffé post hoc tests revealed
a statistically significant effect of time on GSSG 24 h
variations, cosinor detected no statistically significant
rhythm; the waveform of this bimodal rhythm does not
adjust as expected to the cosine wave. In DD, as expected,
the GSH ⁄ GSSG ratio diminished, exhibiting its minimal
peak (at 0400 h); this is coincident after increasing GSSG
levels and decreasing GSH levels.
Figure 4a,b depicts chronograms demonstrating daily
GSH and GSSG oscillations in retina. Reduced glutathione
oscillates with a bimodal statistically significant daily rhythm
(s = 12 h) whose maximal phases take place at 1200 and
2400 h, while GSSG oscillates and exhibits a 24 h significant
daily rhythm whose zenith occurs at 1200 h in mid-photo-
phase. GSH ⁄ GSSG-ratio oscillation shows a maximal value
at 2400 h, when a maximal conversion of GSSG into GSH
occurs, depicting a statistically significant daily oscillation.
After 72 h of darkness, GSSG and GSH concentration
values decrease, exhibiting GSSG statistically significant
bimodal oscillation (s = 11.9 h) and GSH nonsignificant
oscillation by cosinor, although it is statistically significant
by ANOVA (Table 2) and that peaks at the same external
time (0400 h).
In this work, we utilized 24 h glucose hemolymph, retina
and brain variations as daily and circadian metabolic status
crayfish markers, as well as LPO as a biomarker of oxidative
damage in these structures. Figure 5 shows daily variations of
both markers in hemolymph, retina and brain. The chrono-
grams depict a statistically significant daily rhythm of hemol-
ymph glucose concentration (s = 22.8 h), whose zenith is at
16 h, decreasing toward 20 h. Hemolymph glucose concentra-
tion increment coincides with progressively increasing glucose
levels in both retina and brain. These structures present
statistically significant daily uni- and bimodal variations
(s = 24 and 12.8 h, respectively), peaking at 2400 and
1200 h. The glucose increment trend in both structures
coincides with one of the two peaks of statistically significant
retina bimodal daily rhythm LPO (s = 12 h). This rhythm
exhibits two peaks—one at photophase (1600 h) and the other
at scotophase (0400 h); after this second peak, both LPO and
glucose levels decrease. The relationship of glucose and LPO
rhythmic oscillations in the retina and OL-B in DD is shown in
 Photochemistry and Photobiology, 2009, 85 83

Figure 3. Chronograms showing rhythmic changes of reduced (GSH) and oxidized glutathione (GSSG) concentration as well as GSH ⁄ GSSG ratio
calculated from optic lobe-brain (OL-B) samples obtained after light–dark (LD) and dark–dark (DD) conditions. GSH and GSSG concentrations;
(a, c), and GSH ⁄ GSSG ratio (b, d). All data are mean ± standard error (SE) (n = 6). Black and white bars as in Fig. 2. See text for further
information.

Figure 4. Chronograms showing rhythmic changes of reduced (GSH) and oxidized glutathione (GSSG) concentration as well as GSH ⁄ GSSG ratio
calculated from retina samples obtained after 12:12 light–dark cycles (a, b) and dark constant condition.(c, d). All data are mean ± standard error
(SE) (n = 6). Black and white bars as in Fig. 2.
84 Marı́a Luisa Fanjul-Moles et al.
Figure 5. Chronograms showing rhythmic changes in glucose concentration and lipid peroxidation (LPO) in crayfish optic lobe-brain (OL-B) and
retina under 12:12 light–dark cycles (upper panels a, b) and continuous darkness (lower panels c, d) In both graphs hemolymph glucose
concentration data have been plotted for comparison purposes. All data are mean ± standard error (SE) (n = 6). Black and white bars as in
Fig. 2. See text for further information.
Fig. 5c,d. Although under this condition the OL-B complex
shows lower lipid oxidation levels (26.8 nmol mL)1) than the
retina (509.8 nmol mL)1), the corresponding chronograms
depict OL-B and retina lipoperoxidative statistically significant
circadian rhythms (s = 24.4 h, P < 0.05, and s = 21.4 h,
P < 0.05, respectively), whose maximal peaks occur at 2400
and 0800 h. Both glucose and LPO circadian rhythms are anti-
phased, demonstrating a mirror image. Under DD conditions,
these figures depict an evident glucose hemolymph circadian
rhythm with higher amplitude and shorter period (s = 23.5 h,
P < 0.05) than under LD conditions, showing a phase
advance of ca 4 h.
Internal synchrony between rhythms
Figure 6a–c compares the internal phase angle between
acrophase timing of all glutathione rhythms analyzed in this
work with those of hemolymph and liver as analyzed in a
previous work (11). After 72 h of DD, OL-B and retina
glutathione enzymatic rhythms run freely, exhibiting
similar phase angles between them (OL-B w GR-GPx = 1.7 h;
retina w GR-GPx = 1.23 h) but dissimilar ones with their
substrates (OL-B w GPx-GSH = 1.9.0 h, wGR-GSSG = 8.5. h;
retina w GPx-GSH = 8.7 h; wGR-GSSG = 1.23 h). The Rayleigh
test demonstrated statistically significant nonrandom clustering
in OL-B (r = 0.7, P < 0.05), and retina (r = 0.7, P < 0.05),
which means that the vector length differs significantly from
zero, indicating a statistically significant mean direction.
In both structures, nearly all glutathione parameters appear
to reset to LD, advancing or delaying and clustering with a
nonstatistically significant vector in retina (r = 0.5,
P > 0.05), and with a statistically significant vector in OL-B
(r = 0.87, P < 0.05), which demonstrates phase preference
toward dawn (u = 2.8, P < 0.05). Interestingly, under this
condition OL-B and hemolymph GSH and GSSG acrophase
are timed together, showing wGSH-GSSG = 0. In LD retina and
OL-B glucose and LPO acrophase time at the dark phase
showing wLPO-Glu = 3 h (Table 2). After 3 days of darkness,
the phase relationship increased to wLPO-Glu about 12 h in
both. On comparing these values with those obtained in
midgut in a previous work (11) (Fig. 6c), GSH-system behav-
ior in the midgut under DD appears to be more similar to OL-
B than to retina. The V-test demonstrated no statistically
significant differences between midgut and OL-B preferred
mean direction vector, but statistically significant differences
between these organs’ mean direction vectors and that of
retina (u = 2.5, P < 0.05).
DISCUSSION
The results of this study demonstrate statistically significant
bi- and unimodal daily and endogenous rhythms in all GSH-
cycle parameters, substrates and enzymes in OL-B and retina.
The bimodality of some GSH and metabolic rhythms found in
LD could be associated with bimodal characteristics of the
crayfish activity rhythm. Although not recorded in the present

Figure 6. Circular phase maps produced by plotting the acrophases of
glutathione daily and circadian rhythms of optic lobe-brain (OL-B)
(upper graphs), retina (middle graphs), and midgut gland (lower
graphs) under light–dark (LD) (left-hand side) and dark–dark (DD)
(right-hand side) experimental conditions. Each point represents the
acrophase calculated by cosinor analysis. Arrows denote statistically
significant mean direction vectors (Rayleigh test). Midgut gland data
reproduced with permission from Fanjul-Moles et al., Photochemistry
and Photobiology (2003). See text for explanation.
work, this rhythm has been extensively studied (8,9). When the
crayfish is maintained under LD cycles, this rhythm exhibits
two peaks—lights-off endogenous circadian peak and lights-on
exogenous one; thus, in the present work we are unable to
discard a possible influence of locomotor activity on the
rhythmic features of the parameters determined herein.
Figures 2–5 demonstrate the clear bimodal patterns recorded
in LD that change into unimodal circadian patterns after 72 h
of darkness. Although the cosinor detected both bi- and
unimodal patterns under both conditions (Table 2), there is a
clear change of phase that demonstrates the circadian nature
of these rhythms.
Our results indicate an apparent strikingly direct effect of
light on these rhythms that especially in the retina increase
their amplitude greatly under 24 h LD cycles, exhibiting a
greater than three-fold GSH increment. This is a larger
increase than that found in the brain in this work, and in
midgut and hemolymph previously (11). The cyclic luminous
Photochemistry and Photobiology, 2009, 85 85
condition appears to stimulate the entire GSH system in retina
as a response to antagonize the oxidative effect of light on this
organ. Light irradiation producing photo-oxidation is a factor
that determines ROS in different animals (1), as has been
previously demonstrated in crayfish (10). In this work, both
retina and brain showed higher GSH ⁄ GSSG ratio mean values
in LD than in DD. The increase in this parameter coincides
with the photophase, indicating that oxidative stress produced
by light is antagonized by a rapid transformation of GSSG
into GSH. This could be the result of the direct effect of the
LD light cycle on the glutathione system, but especially on GR
activity. Light irradiance could produce an increase in perox-
ides and the subsequent production of GSSG concentration as
a result of increased GPx activity to antagonize this oxidative
stress. This reaction must consume a large amount of the GSH
resulting from a possible direct effect of light on rhythmic GR
activity. The participation of GSH as an antioxidant that
either serves as a substrate for GPx peroxidase to reduce
hydrogen peroxide, or participates directly in different types of
oxidation–reduction reactions (21), one of these involving the
hydroxyl radical, should be particularly important for GSH
participation in the preservation of OL-B, retinal and neural
cells. The hydroxyl radical is one of the most damaging free
radicals, and neurons are particularly vulnerable to free radical
damage (22). Our results are in agreement with some reports
on mammals (23) concerning light-mediated retinal LPO,
potentially an important mechanism of retinal degeneration.
Nonetheless, and albeit that crayfish retina exhibits statistically
significant circadian and daily LPO rhythms, we found an
important increase of this marker in DD, as shown by the
mean (Table 1) and by mesor and amplitude rhythm values
(Table 2). In LD, crayfish retina and brain demonstrate only
mild peroxidation, suggesting that the excitatory effect of light
on the GSH system appears able to antagonize major LPO and
potential retinal degeneration. Photoreceptor membranes, the
site of light absorption, are composed of phospholipids highly
enriched by unsaturated fatty acids (UFA). Interestingly, after
the first 2 h of light exposition visual membranes of the dark-
adapted eye of P. clarkii exhibit a reduction in docosahexa-
enoic acid, one of the longest UFA present in crayfish visual
membranes (24); this decrease is coupled with decreasing LPO
levels (25). This fact could explain the apparent paradoxically
statistical increase in LPO levels in DD with respect to LD.
The resetting effect of light on its circadian clock could allow
crayfish to anticipate changes in the environment for prevent-
ing luminous and metabolic oxidation by means of antioxidant
systems and for preparation prior to the occurrence of both
luminous and metabolic oxidation. It has been proposed that
light—especially UVB-induced ROS—is involved in the acti-
vation of mitogen-activated protein kinase (MAPK) down-
stream antioxidant-response effectors (26), and certain MAPK
signal-regulation regulations by GSH have been described
recently (27). In mammals, much of the GSH synthesis in the
body takes place in hepatocytes and is transported in the
central nervous system via the blood–brain barrier transport-
ers; notwithstanding this, it has been proposed that both
neural and retinal cells are able to synthesize this thiol (28). In
crayfish, GSH synthesis and distribution have been poorly
studied. To our knowledge, this is the first work reporting the
presence of GSH-cycle enzymes and substrates in crustacean
nervous system and retina, although this is not the first work
86 Marı́a Luisa Fanjul-Moles et al.
on crustacean that identifies the relationship between metab-
olism LPO and an antioxidant system. In hepatopancreas and
gills of the estuarine crab Chasmagnatus granulata (29), daily
variations have been identified of metabolic rhythms coincid-
ing with maximal activities of catalase, glutathione-S-trans-
ferase and LPO in the dark phase of the LD cycle. It could be
interesting to identify these enzymatic activities in a new work
based on the findings of the present one, during which we
found two peaks of LPO—one at the photophase, and the
other at the dark phase in a different species.
Although all GSH parameter concentrations and activities
are higher in the midgut and even in the hemolymph (11), the
enhancing effect of light on these parameters in OL-B, and
especially in the retina, is higher than in the midgut. This could
suggest that although these compounds are distributed via the
hemolymph, there is also de novo synthesis through the effect
of light.
In crayfish submitted to constant darkness, the previously
mentioned circadian rhythms persist, but retinal sensitivity
increases, and an increment in time-of-activity (alpha) takes
place (30,31). Thus, we could expect an increase under this
condition of pro-oxidant reactions linked with metabolism and
consequently ROS formation. However, we found no statis-
tically significant differences among glucose concentration in
LD and DD in hemolymph, retina or OL-B that could support
this increasing metabolic flux. We recognize that this param-
eter, although employed here as a metabolic marker, does not
represent a good oxidative-flux marker. It appears plausible
that the higher levels of LPO found under DD in the retina
could be to due to an increase in ROS as a by-product of
respiratory-chain flux that could not be scavenged by GPx, as
indicated by the lower GSH ⁄ GSSG ratio found under this
condition in both structures compared with LD (Table 2).
Despite this decrease, OL-B showed a lower LPO increase than
the retina. This fact may be due both to lower polyunsaturated
acid content in the membranes of these structures compared
with retina, and to the presence of other radical scavengers
such as melatonin, as reported elsewhere in P. clarkii eyestalk
(32). Nevertheless, another possibility could be one proposed
elsewhere (11)—that this may be due to differences in the
glutathione system internal coupling strength of both struc-
tures to the circadian clock. Retina, more susceptible to the
effect of light, resets toward the middle of the subjective day,
anti-phased with LPO and glucose rhythms, of which acro-
phase comprises the middle of the night. Conversely, in OL-B,
GSH ⁄ GSSG increase at the subjective night is coupled with
increasing levels of LPO and glucose concentration, resetting
at dawn. Under darkness, liver and OL-B GSH enzymes and
substrates show similar statistically significant mean vectors
demonstrating a tendency around the same angle while the
retina shows a dissimilar directional preference. This could
suggest higher coupling strength between two possible self-
sustained oscillators (midgut and OL-B) and a passive oscil-
lator, i.e. retina; that appears to show a higher coupling
strength with the zeitgeber, possibly due to a masking effect of
light.
The results of this work indicate the importance of the
relationship between the circadian clock and the redox status
resulting from the metabolism of organisms when they are
exposed to potentially harmful exogenous periodic influences,
such as LD cycles. Endogenous metabolic rhythms in accor-
dance with exogenous rhythms of light irradiation generate
cycles in the redox state. The oxidative stress that occurs can
only be antagonized by the anticipatory adaptive value of the
circadian clock linked with enzymes and scavengers of
antioxidant systems such as that of glutathione. The circadian
clock should allow the crayfish, a nocturnal animal, by means
of activity and metabolism, to synchronize their internal
temporal order to 24 h LD cycles, avoiding oxidative stress in
particular phases of the day–night cycle.
Acknowledgements—We are grateful to Ing. Gerónimo Bello and
Dario Santiago-López, M.Sc., for their technical support in the
implementation of the HPLC technique. We are also in debt to Maggie
Brunner, M.A., for the final English revision of the manuscript. This
work was supported in part by CONACyT México grant 46193-Q and
by PAPIIT IN 208405 and IN 207008 grants. We greatly appreciate the
suggestion and commentaries of the anonymous reviewers that
certainly improved this work.
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