The in Vitro Effect of Metyrapone on Steroid Synthesis in Mice Adrenals at Different Circadian Stages

The circadian rhythm of the in vitro biosynthesis of Cortisol and cortisone in mice adrenals has been documented in the absence and presence of 0.1 μmol metyrapone, an inhibitor of steroid 11β-monooxygenase. After 3 weeks of synchronization with 12 h light: 12 h darkness, adrenalectomy was performed at eight circadian stages: 0, 4, 9, 10, 13, 16, 21, and 22 h after light onset (HALO). Because it has been shown that mice adrenals could convert exogenous 11 -deoxy-cortisol, the synthesis of 11-oxysteroids (Cortisol + cortisone) in adrenal homoge-nates was studied from tritiated precursor. The pattern of steroid synthesis showed a maximum around the end (10 HALO) and a minimum at the beginning of the resting period (0 HALO); the variation was ～ 10%. A similar pattern was observed in the presence of a ～50% inhibiting dose of metyrapone. On the other hand, the percent inhibition of 11-oxysteroids synthesis was greater at the beginning of the resting period (0 HALO) and minimum around the end of the activity span (21 HALO), with an overall variation of 20%. However, the variations were statistically insignificant (unpaired t test).     

Circadian and ultradian (12 h) rhythms of hepatic thiosulfate sulfurtransferase (rhodanese) activity in mice during the first two months of life

Thiosulfate sulfurtransferase (TST) is an important 'enzyme of protection,' that accelerates the detoxification of cyanide, converting it into thiocyanate. The TST physiological rhythm was investigated at wks 2, 4, and 8 of post-natal development (PND) in the mouse. The results revealed a statistically significant gender-related difference, with the highest activity in females, at all the documented PND stages. In the second week of PND (pre-weaning time), the circadian rhythm of the enzyme activity was associated with ultradian components. The prominent circadian rhythm (τ=24 h) peaked at the beginning of the light span, more precisely ∼3 HALO (Hours After Light Onset). A week after weaning (wk 4 of PND), an impairment of the rhythm, with the peak shifted toward the second half of photophase, was recorded. Four to 6 wks later, about wk 8 of PND, the circadian rhythm pattern was stabilized, with its peak then located at the beginning of the dark span (13 HALO). The obtained results showed a 12 h phase-shift of the circadian TST peak time during PND, suggesting that the rhythm stabilization is age-dependent.     

Circadian rhythms of DNA synthesis in nasopharyngeal carcinoma cells

Nasopharyngeal carcinoma (NPC) occurs frequently in southern China. The circadian rhythm of DNA synthesis of a poorly differentiated NPC human cell line (CNE2) was investigated as an experimental prerequisite for designing chrono-chemotherapy schedules for patients with this disease. Twenty-two nude mice with BALB/c background were synchronized alternatively in 12 h of light and 12 h of darkness (LD12:12) for at least 3 wk prior to the transplantation of a CNE2 tumor fragment into each flank (area of ∼2×2 mm²). Ten days later, a tumor sample (area of ∼5 mm²) was obtained at 3, 9, 15, and 21 h after light onset (HALO) alternatively from different sites in each mouse. Single-cell suspensions were prepared and stained with propidium iodide. Cellular DNA content was measured with flow cytometry. Data were analyzed by ANOVA and cosinor methods. The average proportion of tumor cells in G₁, S or G₂-M phase varied according to circadian time with statistical significance. The maximum occurred at 9 HALO for G1, 2 HALO for S and 21 HALO for G₂-M phase cells. The approximate average distribution patterns of G₁ and G₂-M phases of cosine curve was 24 h. This was not the case for S-phase cells, which displayed a bimodal temporal pattern. Inter-individual variability in peak time was large, possibly due to relatively sparse sampling time. Nevertheless, no more than 6% of the time series displayed a maximum at 3 HALO for G₁, 21 HALO for S and 15 HALO for G₂-M. The cell cycle distribution of this human NPC cell line displayed circadian regulation following implantation into nude mice. The mechanisms involved in this rhythm and its relevance to the chrono-chemotherapy of patients deserve further investigation.     

12-hour phase-shift of mice kidney rhodanese (thiosulfate sulfurtransferase) activity in the first two months of life

This study aimed to investigate and compare variation of renal rhodanese activity at 2^nd, 4^th and 8^th weeks of post-natal development (PND) in mice. The enzyme activity increased with age and was higher in females compared to males in all studied groups. Cosinor analysis revealed significant circadian rhythms (with period τ = 24 h) of enzyme activity in both genders with peak time shift during the PND. At the 2^nd week of PND (pre-weaning time), the circadian rhythm peaked at the beginning of light span, more precisely ≅1 HALO (Hours After Light Onset). A week after weaning (4^th week of PND), the peak time was located at the second half of photophase (≅9 HALO) in both genders. Four to six weeks later, about the 8^th week of PND, the circadian peak time was then recorded at ≅13 HALO. These findings suggest that rhodanese level and rhythm stabilization were age-dependent. Moreover, gender-related differences may stimulate discussions on the relationship between renal rhodanese and cyanide sensitivity.     

Circadian rhythm of irinotecan tolerability in mice

The toxicity of irinotecan (CPT-11), a topoisomerase-I inhibitor largely used in cancer patients, was investigated as a function of the circadian time of its administration in mice, with mortality, body weight loss, leukopenia, neutropenia, intestinal lesions, and bone marrow cell cycle phase distribution as end points. Four experiments were performed on a total of 773 male mice standardized with 12h light/12h darkness. Irinotecan was administered daily for 4 or 10 consecutive days (D1-4 and D1-10, respectively, in different experiments) at one of six circadian stages expressed in hours after light onset (HALO). The survival curves differed significantly as a function of the dosage and circadian time of drug administration by the D1-10 schedule, with 70% survival at 7 or 11 HALO and 51% at 19 or 23 HALO (p=0.039 from log rank test). CPT-11 administration at 19 or 23 HALO resulted in (1) greatest mean body weight loss at nadir; (2) most severe colic and bone marrow lesions and/or slowest recovery; and (3) deepest neutropenia nadir and/or slowest hematologic recovery. These circadian treatment time-related differences were statistically validated. The bone marrow cell cycle data revealed a four to eight-fold larger G2-M phase arrest following irinotecan administration at 19 or 23 HALO in comparison to the other times of drug administration, apparently representative of the repair of more extensive DNA damage (p < 0.001 from ANOVA) when the medication was given at these circadian times. Overall, CPT-11 was better tolerated by mice treated during the light (animals' rest) span. The results support the administration of CPT-11 to cancer patients in the second half of the night, during sleep, in order to improve drug tolerability.     

Cyclosporine A dosing-time-dependent effects on plasma creatinine and body weight in male Wistar rats treated for 3 weeks.

Cyclosporine A (CsA) nephrotoxicity was assessed in 120 male Wistar rats (350 +/- 50 g) entrained to a 12-h cycle (light-dark 12:12); plasma creatinine level and body weight were examined in controls and in rats that had been treated daily with oral CsA or vehicle alone (olive oil-ethanol 90:10) for 21 days; daily dosing (40 mg/kg) was at one of six equally spaced given times during the 24-h cycle. The variations observed in both indexes were shown to be circadian dosing stage dependent. Nephrotoxicity was present as early as the third day of treatment with CsA; plasma creatinine level was enhanced by about 50% in rats dosed around the time of the change from darkness to light: at 22 HALO, 146.7 +/- 4.5 mumol/L, against 92.0 +/- 2.8 mumol/L for controls (p less than 0.05); and at 2 HALO, 148.3 +/- 10.0 mumol/L, against 95.0 +/- 4.3 mumol/L for controls (p less than 0.05). Thereafter, a remission episode was observed between days D5-D9. The more drastic effects were seen on days D16 and D21, in animals dosed in the beginning of the dark span (14 HALO): 185 +/- 10 mumol/L for CsA and 98.0 +/- 5.3 mumol/L for controls (p less than 0.01) and, to a lesser extent, in rats treated at the early resting phase (2 HALO): 152.4 +/- 31 mumol/L for CsA and 95.0 +/- 4 mumol/L for controls (p less than 0.05). The normal increase in body weight during the 21-day period (about 14 +/- 8% in controls) was impeded in CsA-administered rats, especially those dosed at the beginning of the activity span (14 HALO) that even suffered weight reduction. Differences in percentages of survivors were noticed, depending on dosing stage. About 40% of the animals in every time CsA-treatment group died, except for those dosed at the end of the resting period (10 HALO), when all animals died. In surviving rats, the cessation of CsA dosing resulted in a reversible effect on the study variables.     

24-hour changes in circulating prolactin, follicle-stimulating hormone, luteinizing hormone, and testosterone in young male rats subjected to calorie restriction

This work analyzes the effect of calorie restriction on the 24 h variation of pituitary-testicular function in young male Wistar rats by measuring the circulating levels of prolactin, follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone. Control animals were provided an equilibrium calorie diet and the experimental animals a calorie-restriction diet equivalent to 66% of food restriction for four weeks starting on day 35 of life. Different groups of control and experimental rats were killed at 6 h intervals around the clock, beginning 1 h after light on (HALO). Compared to the control animals, the mean secretion of prolactin was augmented and that of LH and testosterone decreased in calorie-restricted rats, whereas FSH release remained unchanged. Significant changes in the 24 h secretory pattern of circulating prolactin, LH, and testosterone occurred in the calorie-restricted rats. These include the appearance of a second maximum of plasma prolactin at 21 HALO, blunting of the LH peak seen at 13 HALO, and phase-shift of the testosterone peak from 13 HALO in controls to 17 HALO in calorie-restricted rats. The significant positive correlation between individual LH and testosterone levels found in controls was no longer observed in calorie-restricted rats. Availability of nutrients presumably affects the mechanisms that modulate the circadian variation of the pituitary-gonadal axis in growing male rats.     

Circadian Rhythm of Irinotecan Tolerability in Mice

The toxicity of irinotecan (CPT-11), a topoisomerase-I inhibitor largely used in cancer patients, was investigated as a function of the circadian time of its administration in mice, with mortality, body weight loss, leukopenia, neutropenia, intestinal lesions, and bone marrow cell cycle phase distribution as end points. Four experiments were performed on a total of 773 male mice standardized with 12 h light/12 h darkness. Irinotecan was administered daily for 4 or 10 consecutive days (D1-4 and D1-10, respectively, in different experiments) at one of six circadian stages expressed in hours after light onset (HALO). The survival curves differed significantly as a function of the dosage and circadian time of drug administration by the D1-10 schedule, with 70% survival at 7 or 11 HALO and 51% at 19 or 23 HALO ( p = 0.039 from log rank test). CPT-11 administration at 19 or 23 HALO resulted in (1) greatest mean body weight loss at nadir; (2) most severe colic and bone marrow lesions and/or slowest recovery; and (3) deepest neutropenia nadir and/or slowest hematologic recovery. These circadian treatment time-related differences were statistically validated. The bone marrow cell cycle data revealed a four to eight-fold larger G2-M phase arrest following irinotecan administration at 19 or 23 HALO in comparison to the other times of drug administration, apparently representative of the repair of more extensive DNA damage ( p < 0.001 from ANOVA) when the medication was given at these circadian times. Overall, CPT-11 was better tolerated by mice treated during the light (animals’ rest) span. The results support the administration of CPT-11 to cancer patients in the second half of the night, during sleep, in order to improve drug tolerability.     

Reproductive phase dependent circadian variations of plasma melatonin, testosterone, thyroxine and corticosterone in Indian palm squirrel, Funambulus pennanti

To explain the complex mechanism of environmental influence along with internal hormonal (factors) milieu on daily variations in the circulating levels of melatonin, testosterone, thyroxine and corticosterone were analyzed with the help of inferential statistics (Cosinor rhythmometry) in a seasonally breeding tropical rodent, F. pennanti during the reproductively active (RAP) and inactive phases (RIP). Plasma melatonin, thyroxine and corticosterone levels exhibited a significant circadian oscillation during both the active and inactive phases of the annual reproductive cycle. Melatonin showed higher amplitude during RIP in the circulating plasma. Testosterone presented a peak level during evening hours (16:00 - 18:00 h) during RAP only. The phase of thyroxine was noted ∼09:76 h and ∼10.35 h during active and inactive phases, respectively. Corticosterone showed a peak level at ∼12.00 h during both phases of the reproductive cycle. Further, in this tropical rodent, the minimum difference in photoperiod (∼3 - 4 hours) and maximum variation in temperature (max. 18°C - min. 10°C during RIP and max. 45°C - min. 32°C during RAP) and humidity (85% during RIP and 35% during RAP) regulated the diurnal rhythm of circulating melatonin circadian rhythm by ∼1 hour phase advance during RIP. In conclusion, the studied hormonal rhythms may be part of an integrative system to coordinate reproduction and physiological processes successfully with environmental factors.     

Chronotoxicology of florfenicol

Sixty 3-month-old homozygote male mice were studied for circadian rhythmicity in the toxicity of florfenicol overdose. Animals were kept under a regimen of 12h light, 12h darkness (12:12 LD) with food and water available ad libitum. The LD50 (median lethal) dose was determined in a preliminary experiment and was administered to groups of 10 mice at six different clock times (hours) after light onset (HALO): 0, 4, 8, 12, 16, and 20 HALO. Cosinor analysis verified a statistically significant (P < .04) circadian rhythm in the toxic effect (mortality) of florfenicol. Mortality was greatest when the drug was injected 4h after the commencement of the activity span (16 HALO) and least when injected 4h after the start of the diurnal rest span (4 HALO). Mortality was 2.5 times greater when drug injection was given at 16 HALO than at 4 HALO.     

Effects of photophase and altitude on oviposition rhythm of the himalayan strains of Drosophila ananassae

The effects of varying photophase and altitude of origin on the phase angle difference (Ψ) of the circadian rhythm of oviposition during entrainment to light-dark (LD) cycles and the aftereffects of such photophases on the period of the free-running rhythm (τ) in constant darkness (DD) were evaluated in two Himalayan strains of Drosophila ananassae, the high-altitude (HA) strain from Badrinath (5,123 m above sea level=ASL) and the low-altitude (LA) strain from Firozpur (179 m ASL). The Ψ (i.e., the hours from lights-on of the LD cycle to oviposition median) of both strains was determined in LD cycles in which the photophase at 100 lux varied from 6 to 18 h/24 h. The HA strain was entrained by all LD cycles except the one with 6 h photophase in which it was weakly rhythmic, but the LA strain was entrained by only three LD cycles with photophases of 10, 12, and 14 h, but photophases of 6, 8, 16, and 18 h rendered it arrhythmic. Lights-off transition of LD cycles was the phase-determining signal for both strains as oviposition medians of the HA strain occurred∼6 h prior to lights-off, while those of the LA strain occurred∼1 h after lights-off. The Ψ of the HA strain increased from∼2 h in 8 h photophase to∼11 h in 18 h photophase, while that of the LA strain increased from∼11 h in 10 h photophase to∼15 h in 14 h photophase. The aftereffects of photophase of the prior entraining LD cycles on τ in DD were determined by transferring flies from LD cycles to DD. The τ of the HA strain increased from∼19 to∼25 h when transferred to DD from LD 8:16 and LD 18:6 cycles, respectively, whereas the τ of the LA strain increased from∼26 to∼28 h when transferred to DD from LD 10:14 and LD 14:10 cycles, respectively. Thus, these results demonstrate that the photophases of entraining LD cycles and the altitude of origin affected several parameters of entrainment and the period of the free-running rhythm of these strains.     

The photoperiodic entrainment and induction of the circadian clock regulating seasonal responses in the migratory blackheaded bunting (Emberiza melanocephala)

The properties of the circadian photoperiodic oscillator have been investigated in detail only in the Japanese quail. While the study of the quail is clearly very important, one cannot simply assume that other species, especially passerines that seem to have a different circadian organization than quail, function the same way. The current set of experiments was conducted to understand the entrainment and photoinduction of the circadian photoperiodic oscillator in a passerine species, the blackheaded bunting (Emberiza melanocephala). The experimental paradigm used skeleton photoperiods with two light periods, the first called the “entraining light pulse” (E-pulse) and the second called the “inducing light pulse” (I-pulse). Three experiments were performed on photosensitive male birds (N=6-8/group). Experiment 1 investigated the effects of the temporal relationship between E- and I-pulses on photoperiodic induction. Buntings entrained to 8h:16h L:D for 4 wk were released into constant dim light (LL_dim, ∼1 lux). Beginning on subjective day 8, they received for 8 wk, E- and I-pulses only at alternate cycles. While I-pulse was 1 h and always began at zt 11.5, E-pulse varied in duration and timing (the 1h E-pulse beginning either at zt 0, zt 5, or zt 9, the 4h one beginning at zt 0 or zt 6, and the 10h one at zt 0; zeitgeber time 0=time of lights-on under 8h:16h L:D prior to release into LL_dim). A photoperiodic response was induced only when the E-pulse began at zt 0, and thus the beginning of E- and I-pulses were separated by 11.5 h. Experiment 2 determined whether the duration of the E-pulse influences the position of the photoinducible phase (φi) of the circadian photoperiodic oscillator. Birds were entrained to 1h:23h L:D or 10h:14h L:D for 2 wk, and then exposed to 1h I-pulse at zt 11.5, zt 15, or zt 18.5 for another 8 wk. Photoperiodic induction occurred at all 3 zts in birds entrained to 10 h but only at zt 11.5 in birds entrained to 1 h, which infers the circadian rhythm of photoinducibility (CRP) in buntings was re-entrained when I-pulse fell at zt 15 and after. The last experiment examined the possibility of the re-entrainment of the CRP to light pulses falling at zt 15 and after. Birds received 1h I-pulse for 8 wk at zt 15 following 2 wk of 2.5h:21.5h L:D or 3.5h:20.5h L:D, or at zt 21.5 or zt 22.5 following 2 wk of 10h:14h LD. Photoperiodic induction was consistent with the hypothesis of the re-entrainment of the CRP under these light-dark cycles. The I-pulse appeared to be interpreted as a “new dawn”, and so the photoperiodic induction was determined by the coincidence of φi with the E-pulse. These results suggest a phase-dependent action of light on the circadian oscillator regulating photoperiodic responses in the blackheaded bunting. This could be a useful strategy for a photoperiodic species to regulate its seasonal responses in nature.     

24-Hour Changes in Circulating Prolactin,Follicle-Stimulating Hormone,Luteinizing Hormone,and Testosterone in Young Male Rats Subjected to Calorie Restriction

This work analyzes the effect of calorie restriction on the 24 h variation of pituitary-testicular function in young male Wistar rats by measuring the circulating levels of prolactin, follicle-stimulating hormone (FSH), luteinizing hormone (LH), and testosterone. Control animals were provided an equilibrium calorie diet and the experimental animals a calorie-restriction diet equivalent to 66% of food restriction for four weeks starting on day 35 of life. Different groups of control and experimental rats were killed at 6 h intervals around the clock, beginning 1 h after light on (HALO). Compared to the control animals, the mean secretion of prolactin was augmented and that of LH and testosterone decreased in calorie-restricted rats, whereas FSH release remained unchanged. Significant changes in the 24 h secretory pattern of circulating prolactin, LH, and testosterone occurred in the calorie-restricted rats. These include the appearance of a second maximum of plasma prolactin at 21 HALO, blunting of the LH peak seen at 13 HALO, and phase-shift of the testosterone peak from 13 HALO in controls to 17 HALO in calorie-restricted rats. The significant positive correlation between individual LH and testosterone levels found in controls was no longer observed in calorie-restricted rats. Availability of nutrients presumably affects the mechanisms that modulate the circadian variation of the pituitary-gonadal axis in growing male rats.     

Circadian Dependent Effect of Epidermal Growth Factor, Insulin and Glucagon on Hepatic Pyruvate Kinase and Malic Enzyme of Mice

The influence of epidermal growth factor (EGF), 0.75 μg g^-1; insulin, 1.5 μg g^-1; glucagon, 1.25 ygg^-1 and their combinations on the activities of hepatic pyruvate kinase (PK) and malic enzymes (ME) was monitored. Male CD2F₁ mice were treated toward the end of the light or dark periods, 9 or 23 /tours after /ights on (9 or 23 HALO), and subgroups of six mice were killed at 4,8 or 12 hr post-treatment. PK and ME activities from control mice were well characterized by cosine curves. The PK activity was maximal when ME activity was minimal at the transition from light to dark (9 HALO plus 4 hr) and PK was at a minimum when ME was highest (23 HALO plus 4 hr). Both enzymes were influenced by at least one peptide hormone, and the effects were strongly circadian -stage dependent. The only effect attributed to EGF was an increase of PK activity (23%) 12 hr after injection at 23 HALO. PK activity was increased by insulin (23%) at 23 HALO (4 hr after injection), but not at 9 HALO, and decreased (17%) by glucagon 12 hr after injection at 9 HALO. Several reductions in PK activity in response to various combinations of peptides were observed, and appeared to be caused by glucagon but influenced by insulin. The activity of ME was decreased (33%) in response to insulin 4 hr after injection at 23 HALO but not at 9 HALO and increased (60-70%) by glucagon alone or in combinations with insulin or EGF, or both, at 4 hr after injection at 9 HALO but not at 23 HALO. In general, when ME activity was altered by either insulin or glucagon, PK activity was also altered in the opposite direction, and the effects of glucagon were opposed by insulin.     

CHRONOTOXICOLOGY OF FLORFENICOL

Sixty 3-month-old homozygote male mice were studied for circadian rhythmicity in the toxicity of florfenicol overdose. Animals were kept under a regimen of 12h light, 12h darkness (12:12 LD) with food and water available ad libitum. The LD₅₀ (median lethal) dose was determined in a preliminary experiment and was administered to groups of 10 mice at six different clock times (hours) after light onset (HALO): 0, 4, 8, 12, 16, and 20 HALO. Cosinor analysis verified a statistically significant (P <. 04) circadian rhythm in the toxic effect (mortality) of florfenicol. Mortality was greatest when the drug was injected 4h after the commencement of the activity span (16 HALO) and least when injected 4h after the start of the diurnal rest span (4 HALO). Mortality was 2.5 times greater when drug injection was given at 16 HALO than at 4 HALO. (Chronobiology International, 18(3), 567–572, 2001)     

Orcadian Rhythm of Trehalose in the Face Fly Musca Autumnalis De Geer

Trehalose levels were determined over two 24 hr spans in groups of face fly adults 3-4 days after emergence from the puparium. Face fly pupae were placed in rearing chambers at 27° C in a staggered light-dark regimen, LD 16:8, so that at a given clock hour, samples could be obtained at several different hours after lights on (HALO). Trehalose was determined in hemolymph collected from a puncture in the intersegmental membrane of the abdomen. Treated hemolymph samples were passed through a Bio-Rad Amino S-S disaccharide column and a Waters 410 refractive index detector was used to differentiate among sugars. The circadian acrophase derived by cosinor analysis in hemolymph trehalose (when the values were 25.49 and 26.86μg/μ¹ on the first and second days respectively) occurred at -226° (ca 15 HALO) and the bathyphase at 24 HALO. The mesor = 11.82μg/μ¹ trehalose, the amplitude = 8.57/μg/μ¹ trehalose and the P-value for presence of a rhythm was 0.003. Based on these data, differences between control and test flies in a bioassay of hypertrehalosemic activity would be most easily observed at 0-8 HALO, while exogenous hypotrehalosemic activity would be best assayed at 12-20 HALO.     

Controlled exposure to light and darkness realigns the salivary cortisol rhythm in night shift workers

The efficacy of a light/darkness intervention designed to promote circadian adaptation to night shift work was tested in this combined field and laboratory study. Six full-time night shift workers (mean age ± SD:37.1 ± 8.1 yrs) were provided an intervention consisting of an intermittent exposure to full-spectrum bright white light (∼2000 lux) in the first 6 h of their 8 h shift, shielding from morning light by tinted lenses (neutral gray density, 15% visual light transmission), and regular sleep/darkness episodes in darkened quarters beginning 2 h after the end of each shift. Five control group workers (41.1 ± 9.9 yrs) were observed in the presence of a regular sleep/darkness schedule only. Constant routines (CR) performed before and after a sequence of ∼12 night shifts over 3 weeks revealed that treatment group workers displayed significant shifts in the time of peak cortisol expression and realignment of the rhythm with the night-oriented schedule. Smaller phase shifts, suggesting an incomplete adaptation to the shift work schedule, were observed in the control group. Our observations support the careful control of the pattern of light and darkness exposure for the adaptation of physiological rhythms to night shift work.     

Relationship of atypical melatonin rhythm with two circadian clock outputs in B6D2F(1) mice

Circadian rhythms in body temperature, locomotor activity, and the circadian changes of plasma and pineal melatonin content were investigated in B6D2F(1) mice synchronized by 12 h of light and 12 h of darkness. During 8 wk continuous recording, activity and temperature displayed a marked stable and reproducible circadian rhythm, with both peaks occurring near the middle of darkness. Both 24- and 12-h rhythmic components were also significantly detected. Mean plasma melatonin concentration rose steadily during the light span and reached a maximum (30.6 +/- 10.0 pg/ml) at 11 h after light onset (HALO), then gradually decreased after the onset of darkness to a nadir (4.7 +/- 0.4 pg/ml) at 20 HALO. Mean pineal content followed a pattern parallel to that of plasma concentration (peak at 11 HALO: 17.7 +/- 1.0 pg/gland; trough at 17 HALO: 4.7 +/- 1.0 pg/gland). In addition, a second sharp peak was observed at 21 HALO (20.2 +/- 3.5 pg/gland). Plasma and pineal contents displayed large and statistically significant circadian changes, with a composite rhythm of period (24 + 12 h). This mouse model has predominant production and secretion of melatonin during the day. This possibly contributes to a similar coupling between chronopharmacology mechanisms and the rest-activity cycle in these mice and in human subjects.     

Pharmacological effects of vinorelbine on body temperature and locomotor activity circadian rhythms in mice

The effects of vinorelbine (VRL) on the circadian rhythms in body temperature and locomotor activity were investigated in unrestrained B6D2F₁ mice implanted with radio-telemetry transmitters. A single intravenous VRL dose (24 or 12 mg/kg) was given at 7 h after light onset (HALO), a time of high VRL toxicity, and resulted in transient suppression of temperature and activity circadian rhythms in mice kept in light-dark (LD) 12h:12h. Such suppression was dose-dependent. It occurred within 1-5 d after VRL dosing. Recovery of both rhythms was partially complete within 5 d following the high dose and within 2 or 3 d after the low dose and was not influenced by suppression of photoperiodic synchronization by housing in continuous darkness. Moreover, VRL induced a dose-dependent relative decrease in amplitude and phase shift of the temperature circadian rhythm. The mesor and amplitude of the activity rhythm were markedly reduced following the VRL administration. The relevance of VRL dosing time was studied in mice housed in LD 12h:12h. Vinorelbine was injected weekly (20 mg/kg/injection) for 3 wk at 6 or 18 HALO. Vinorelbine treatment ablated the rest-activity and temperature rhythms 3-6 d after each dose, with fewer alterations after VRL dosing at 18 HALO compared to 6 HALO, especially for the body temperature rhythm. There was at least partial recovery 1 wk after dosing, suggesting the weekly schedule of drug treatment is acceptable for therapeutic purposes. Our findings demonstrate that VRL can transiently, yet profoundly, alter circadian clock function. Vinorelbine-induced circadian dysfunction may contribute to the toxicokinetics of this and possibly other anticancer drugs.     

Circadian Reactivity Rhythm of Rat Gastric Mucosa to Restraint-Cold Stress and Indomethacin: Temporal Variation in the Protective Effect of Iloprost

In this study, the time-dependent ulcerogenic effects of restraint-cold stress and indomethacin on the gastric mucosa and the temporal variation in the protective effect of iloprost, a synthetic stable analog of prostacyclin, were investigated in rats synchronized to 12h light and 12h darkness, lights on at 08:00. The severity of gastric ulceration produced by either stress or indomethacin showed marked circadian variation; it was greatest at 11 HALO (hours after lights on) for restraint-cold stress and at 23 HALO for indomethacin. The severity of the induced ulcerogenesis was least at 7 HALO for both stimuli. The protective effect of iloprost against restraint-cold stress was most prominent at 15 HALO and 19 HALO with an approximately 80% protection score. On the other hand, pretreatment with iloprost reduced the indomethacin-induced mucosal injury only at 23 HALO. The circadian variation in the effect of iloprost and in the rhythmic modalities of these two experimental ulcer models are indicative of differences in their underlying mechanisms. In experimental models of ulceration, the circadian time of application of the ulcerogenic stimulus must be considered as an important experimental factor. Moreover, the protective effectiveness of antiulcer drugs can express time-dependent differences and must also be taken into account in investigative research. (Chronobiology International, 14(6), 575–583, 1997)