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78
Photochemistry and Photobiology, 2009, 85 79
LD and DD in retina and OL-B, a Student’s t-test was performed. reductase and GPx also demonstrated lower activity than that
Significant clustering of phases in both LD and DD were determined reported in the same work for hepatic tissue as follows: LD
using circular statistics (Rayleigh and V-tests). Both tests examine
whether there is statistical evidence of directional phase preference in
retina GR = 11.7 ± 2.5 lM NADPH ox g)1 prot min)1;
the population from which the sample is drawn (20). 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
RESULTS GR = 37.4 ± 4.9 lM NADPH ox g)1 prot min)1; GPx =
Analysis of crayfish retina and OL-B complex extracts revealed 8.3 ± 1.9 lM NADPH ox g)1 prot min)1; DD OL-B,
the presence of two substances that oxidized at 1.35 V and GR = 6.7 ± 1.3 lM NADPH ox g)1 prot min)1; and GPx =
eluted at the same time as the GSH and GSSG standard (mean 15.33 ± 3.8 lM NADPH ox g)1 prot min)1. The amount
elution time = 4 and 8 min, respectively). In addition, a single and activity of all glutathione system parameters changed with
symmetrical and additive peak was observed for both sub- the luminous condition. The Student’s t-test revealed signifi-
stances when GSH and GSSG external standards were added cant differences in all parameters except GSSG and GR
to acid extracts in each injection (Fig. 1). The voltage– between LD and DD in brain and retina, respectively (Table 1).
amperage curve generated for GSH and GSSG standards
and presumptive peaks in the sample were also identical. Based
Daily and circadian rhythm determination
on these criteria, these two peaks were identified as GSH and
GSSG. Chronograms showing GR and GPx temporal activity in
Results of the current study demonstrate the presence of all OL-B are depicted in Fig. 2a,b. In LD, the GR activity peak is
GSH-system components in crayfish retina and OL-B. These at 0800 h, apparently increasing immediately after lights on at
structures show lower GSH and GSSG concentration values 0700 h, with a second peak appearing at 2000 h after lights off
than those reported for hemolymph and midgut in the same at 1900 h. Cosinor analysis revealed a GR bimodal significant
species (10) as follows: LD retina GSH = 42.6 ± 3.7 lM and daily rhythm with a 12 h period value (Table 2). Both peaks
GSSG = 8.6 ± 1.0 lM; DD retina GSH = 14.96 ± 1 lM are coincident with maximal peaks of GPx bimodal activity,
and GSSG = 6.12 ± 0.8 lM; LD OL-B GSH = 26.4 ± although GPx showed lower average activity throughout 24 h
2.0 lM and GSSG = 2.35 ± 0.2 lM; and DD OL-B GSH = (M = 8.6) than GR (M = 36.9 ± 3.8) (Table 2). After 72 h
12.5 ± 0.8 lM and GSSG = 7.3 ± 1.2 lM. Glutathione of darkness, the mean GPx activity level increases (M = 16.1),
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.
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.
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 by ANOVA (Table 2) and that peaks at the same external
a statistically significant effect of time on GSSG 24 h time (0400 h).
variations, cosinor detected no statistically significant In this work, we utilized 24 h glucose hemolymph, retina
rhythm; the waveform of this bimodal rhythm does not and brain variations as daily and circadian metabolic status
adjust as expected to the cosine wave. In DD, as expected, crayfish markers, as well as LPO as a biomarker of oxidative
the GSH ⁄ GSSG ratio diminished, exhibiting its minimal damage in these structures. Figure 5 shows daily variations of
peak (at 0400 h); this is coincident after increasing GSSG both markers in hemolymph, retina and brain. The chrono-
levels and decreasing GSH levels. grams depict a statistically significant daily rhythm of hemol-
Figure 4a,b depicts chronograms demonstrating daily ymph glucose concentration (s = 22.8 h), whose zenith is at
GSH and GSSG oscillations in retina. Reduced glutathione 16 h, decreasing toward 20 h. Hemolymph glucose concentra-
oscillates with a bimodal statistically significant daily rhythm tion increment coincides with progressively increasing glucose
(s = 12 h) whose maximal phases take place at 1200 and levels in both retina and brain. These structures present
2400 h, while GSSG oscillates and exhibits a 24 h significant statistically significant daily uni- and bimodal variations
daily rhythm whose zenith occurs at 1200 h in mid-photo- (s = 24 and 12.8 h, respectively), peaking at 2400 and
phase. GSH ⁄ GSSG-ratio oscillation shows a maximal value 1200 h. The glucose increment trend in both structures
at 2400 h, when a maximal conversion of GSSG into GSH coincides with one of the two peaks of statistically significant
occurs, depicting a statistically significant daily oscillation. retina bimodal daily rhythm LPO (s = 12 h). This rhythm
After 72 h of darkness, GSSG and GSH concentration exhibits two peaks—one at photophase (1600 h) and the other
values decrease, exhibiting GSSG statistically significant at scotophase (0400 h); after this second peak, both LPO and
bimodal oscillation (s = 11.9 h) and GSH nonsignificant glucose levels decrease. The relationship of glucose and LPO
oscillation by cosinor, although it is statistically significant 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 In both structures, nearly all glutathione parameters appear
shows lower lipid oxidation levels (26.8 nmol mL)1) than the to reset to LD, advancing or delaying and clustering with a
retina (509.8 nmol mL)1), the corresponding chronograms nonstatistically significant vector in retina (r = 0.5,
depict OL-B and retina lipoperoxidative statistically significant P > 0.05), and with a statistically significant vector in OL-B
circadian rhythms (s = 24.4 h, P < 0.05, and s = 21.4 h, (r = 0.87, P < 0.05), which demonstrates phase preference
P < 0.05, respectively), whose maximal peaks occur at 2400 toward dawn (u = 2.8, P < 0.05). Interestingly, under this
and 0800 h. Both glucose and LPO circadian rhythms are anti- condition OL-B and hemolymph GSH and GSSG acrophase
phased, demonstrating a mirror image. Under DD conditions, are timed together, showing wGSH-GSSG = 0. In LD retina and
these figures depict an evident glucose hemolymph circadian OL-B glucose and LPO acrophase time at the dark phase
rhythm with higher amplitude and shorter period (s = 23.5 h, showing wLPO-Glu = 3 h (Table 2). After 3 days of darkness,
P < 0.05) than under LD conditions, showing a phase the phase relationship increased to wLPO-Glu about 12 h in
advance of ca 4 h. 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-
Internal synchrony between rhythms
B than to retina. The V-test demonstrated no statistically
Figure 6a–c compares the internal phase angle between significant differences between midgut and OL-B preferred
acrophase timing of all glutathione rhythms analyzed in this mean direction vector, but statistically significant differences
work with those of hemolymph and liver as analyzed in a between these organs’ mean direction vectors and that of
previous work (11). After 72 h of DD, OL-B and retina retina (u = 2.5, P < 0.05).
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
DISCUSSION
substrates (OL-B w GPx-GSH = 1.9.0 h, wGR-GSSG = 8.5. h; The results of this study demonstrate statistically significant
retina w GPx-GSH = 8.7 h; wGR-GSSG = 1.23 h). The Rayleigh bi- and unimodal daily and endogenous rhythms in all GSH-
test demonstrated statistically significant nonrandom clustering cycle parameters, substrates and enzymes in OL-B and retina.
in OL-B (r = 0.7, P < 0.05), and retina (r = 0.7, P < 0.05), The bimodality of some GSH and metabolic rhythms found in
which means that the vector length differs significantly from LD could be associated with bimodal characteristics of the
zero, indicating a statistically significant mean direction. crayfish activity rhythm. Although not recorded in the present
Photochemistry and Photobiology, 2009, 85 85
on crustacean that identifies the relationship between metab- dance with exogenous rhythms of light irradiation generate
olism LPO and an antioxidant system. In hepatopancreas and cycles in the redox state. The oxidative stress that occurs can
gills of the estuarine crab Chasmagnatus granulata (29), daily only be antagonized by the anticipatory adaptive value of the
variations have been identified of metabolic rhythms coincid- circadian clock linked with enzymes and scavengers of
ing with maximal activities of catalase, glutathione-S-trans- antioxidant systems such as that of glutathione. The circadian
ferase and LPO in the dark phase of the LD cycle. It could be clock should allow the crayfish, a nocturnal animal, by means
interesting to identify these enzymatic activities in a new work of activity and metabolism, to synchronize their internal
based on the findings of the present one, during which we temporal order to 24 h LD cycles, avoiding oxidative stress in
found two peaks of LPO—one at the photophase, and the particular phases of the day–night cycle.
other at the dark phase in a different species.
Although all GSH parameter concentrations and activities Acknowledgements—We are grateful to Ing. Gerónimo Bello and
are higher in the midgut and even in the hemolymph (11), the Dario Santiago-López, M.Sc., for their technical support in the
enhancing effect of light on these parameters in OL-B, and implementation of the HPLC technique. We are also in debt to Maggie
especially in the retina, is higher than in the midgut. This could Brunner, M.A., for the final English revision of the manuscript. This
suggest that although these compounds are distributed via the work was supported in part by CONACyT México grant 46193-Q and
hemolymph, there is also de novo synthesis through the effect by PAPIIT IN 208405 and IN 207008 grants. We greatly appreciate the
suggestion and commentaries of the anonymous reviewers that
of light.
certainly improved this work.
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
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