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)     

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.     

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 time-dependent differences in murine tolerance to the antihistaminic agent loratadine

Loratadine is a second-generation histamine H(1)-receptor antagonist used in the treatment of allergic diseases. The aim of the study was to assess whether lethal toxicity and motor incoordination (neurotoxicity) of loratadine is circadian rhythm-dependent. A total of 210 male Swiss mice, aged 10 wk, were synchronized for 3 wk to 12 h light (rest span)/12 h dark (activity span). The drug was administered per os. The choice of the sublethal (TD(50) = 82 mg/kg body weight) and the lethal (LD(50) = 4 g/kg body weight) dosage was based on preliminary studies. Each of these two doses was administered to comparable groups of animals at six different circadian time points (1, 5, 9, 13, 17, and 21 Hours After Light Onset [HALO]). The survival duration was dosing time-dependent (chi(2) = 16.96; p < 0.001). Drug dosing at 17 HALO resulted in best (67%) survival rate; whereas, dosing at 9 HALO resulted in poorest (21%) survival rate. Cosinor analyses (with a trial period tau = 24 h) validated a statistically significant circadian rhythm in survival rate (p < 0.04) with an acrophase (peak time ? of best tolerance to loratadine) being at 17.5 HALO +/- 4.65 h. Troughs of motor incoordination were located at the administration times of 5 and 17 HALO (60% and 32% of animals affected, respectively), whereas peaks were located at 9 and 21 HALO (87% and 68% of animals affected, respectively). The 24 h mean of the motor incoordination was 61%, the mean proportion of animals affected by the treatment for the six different circadian times studies. The extent of this neurotoxic effect varied as a function of loratadine dosing time (p < 0.001). A statistically significant ultradian component rhythm (p < 0.01) with a trial period tau = 12 h was also validated. The obtained results show that the dosing time of loratadine at the mid-activity (dark) span seems to be optimal, since it corresponds to the longest (21 vs. 12 days) survival span and to least neurotoxicity.     

Circadian rhythms in toxic effects of the serotonin antagonist ondansetron in mice

The aim of the study was to learn whether the lethal and the motor incoordination (ataxia) side effect of ondansetron (Zophren) administration is dosing-time dependent. Ondansetron is a serotonin 5-HT3 receptor antagonist used primarily to control nausea and vomiting arising from cytotoxic chemo- and radiotherapy. A total of 210 male Swiss mice 10 to 12 weeks of age were synchronized for 3 weeks by 12 h light (rest span)/12 h dark (activity span). Different doses of ondansetron were injected intraperitoneally (i.p.) at fixed times during the day to determine both the sublethal (TD50) and lethal (LD50) doses, which were, respectively, 3.7 +/- 0.6 mg/kg and 4.6 +/- 0.5 mg/kg. In the chronotoxicologic study a single dose of ondansetron (3.5 mg/kg, i.p.) was administered to different and comparable groups of animals at four different circadian stages [1, 7, 13, and 19 h after light onset (HALO)]. The lethal toxicity was statistically significantly dosing time-dependent (chi2 = 21.51, p < 0.0001). Drug dosing at 1 HALO resulted in 100% survival rate whereas drug dosing at 19 HALO was only one-half that (52%). Similarly, lowest and highest ataxia occurred when ondansetron was injected at 1 and 19 HALO, respectively (chi2 = 22.24, p < 0.0001). Effects on rectal temperature were also dosing-time related (Cosinor analysis, p < 0.0001). The characteristics of the waveform describing the temporal patterns differed between the studied variables, e.g., lethal toxicity and survival rate showing two peaks and rectal temperature showing one peak in the 24 h time series waveform pattern. Cosinor analysis also revealed a statistically significant ultradian (tau = 8 h) rhythmic component in the considered variables. Differences in curve patterns in toxicity elicited by ondansetron on a per end point basis are hypothesized to represent the phase relations between the identified 24 h and 8 h periodicities.     

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.     

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).     

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 variation in hepatic toxicity of the immunosuppressive agent “Mycophenolate Mofetil” in rats

Immunosuppressive drugs may have varying toxicity or efficacy depending on the administration time. This study investigates whether the liver toxicity of the mycophenolate mofetil (MMF) varies according to the circadian dosing-time in rats. 300 mg/kg of MMF was injected by intraperitonal route to different groups of animals at four different circadian stages (1, 7, 13, and 19 h after light onset, HALO). Biochemical variable (transaminase, alkaline phosphatase) and histopathological examinations on liver section were performed. The results obtained showed that MMF treatment induced hepato-toxicity depending on the circadian time. A severe toxicity in the liver was observed when the drug was injected at 7 HALO. The data obtained indicate that the maximum of hepato-toxicity is observed when MMF was injected in the middle of the light-rest span of rats which is physiologically analogous to the end of the activity of the diurnal phase in human patients.     

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.     

Circadian time-effect of orally administered loratadine on plasma pharmacokinetics in mice

Little is known about the chronopharmacokinetics of loratadine, a long-acting tricyclic antihistamine H(1) widely used in the treatment of allergic diseases. Hence, the pharmacokinetics of loratadine and its major metabolite, desloratadine, were investigated after a 20 mg/kg dose of loratadine had been orally administered to comparable groups of mice (n=33), synchronized for three weeks to 12 h light (rest span)/12 h dark (activity span). The drug was administered at three different circadian times (1, 9, and 17 h after light onset [HALO]). Multiple blood samples were collected over 48 h, and plasma concentrations of loratadine and desloratadine were determined by high performance liquid chromatography. There were no significant differences in T(max) of loratadine and desloratadine between treatment-time different groups. However, the elimination half-life (t1/2) of the parent compound and its metabolite was significantly longer (p<0.01) following administration at 9 HALO (t1/2 loratadine and desloratadine 5.62 and 4.08 h at 9 HALO vs. 4.29 and 2.6 h at 17 HALO vs. 3.26 and 3.27 at 1 HALO). There were relevant (p<0.05) differences in C(max) between the three treated groups for loratadine and desloratadine; 133.05+/-3.55 and 258.07+/-14.45 ng/mL at 9 HALO vs. 104.5+/-2.61 and 188.62+/-7.20 ng/mL at 1 HALO vs. 94.33+/-20 and 187.75+/-10.79 ng/mL at 17 HALO. Drug dosing at 17 HALO resulted in highest loratadine and desloratadine total apparent clearance values: 61.46 and 15.97 L/h/kg, respectively, whereas loratadine and desloratadine clearances (CL) were significantly slower (p<0.05) at the other administration times (loratadine and desloratadine CL was 57.3 and 14.22 L/h/kg at 1 HALO vs. 43.79 and 12.89 L/h/kg at 9 HALO, respectively). The area under the concentration-time curve (AUC) of loratadine and desloratadine was significantly (p<0.05) greater following drug administration at 9 HALO (456.75 and 1550.57 (ng/mL) . h, respectively); it was lowest following treatment at 17 HALO (325.39 and 1252.53 (ng/mL) . h, respectively). These pharmacokinetic data indicate that the administration time of loratadine significantly affected its pharmacokinetics: the elimination of loratadine and its major metabolite desloratadine.     

Circadian time-dependent hepatic and renal toxicities to valproic acid in mice

This study aims to investigate whether hepatic and renal valproic acid (VPA) toxicities varied according to the dosing time in the 24-h scale in mice. VPA was administered by i.p. route to different groups of animals at four different circadian stages (1, 7, 13, and 19 h after light onset (HALO)). Biochemical study and histopathological examinations on liver and kidney sections were performed. The results showed that the hepatic and renal toxicity induced by VPA was time related. Animals treated at 19 HALO showed vacuolar degenerative changes, congestions, and inflammatory areas on liver parenchyma. Lesions within proximal tubules were observed in the kidney in groups treated at 19 HALO. The largest increases in alanine aminotransferase, alkaline phosphatase and plasma creatinine activities were also observed at 19 HALO. The obtained data indicate that the optimal hepatic and renal tolerance is observed when VPA was injected in the middle of the light-rest span of mice.     

Malondialdehyde content and circadian variations in brain, kidney, liver, and plasma of mice

In aerobic organisms, the use of oxygen (O(2)) to produce energy is associated with the production of Reactive Oxygen Species (ROS), which reacts with biological molecules to produce oxidized metabolites such as malondialdehyde (MDA). This experiment focused on male Swiss mice 12 weeks of age synchronized for 3 weeks by the 12 h light (rest)/12 h dark (activity) span. Different and comparable groups of animals (n=10) were sacrificed at six different circadian stages: 1, 5, 9, 13, 17, and 21 h after light onset (HALO). The 24 h mean MDA level varied among organs of mice in non-stress conditions and was comparable in brain and liver but lower than in kidney. As the MDA 24 h status constitutes only a part of ROS damages in sites differing by their oxygen use, lipid composition, and detoxification capacity, the temporal patterns of their MDA content were comparatively studied in relationship to the animal rest-activity cycle. The results revealed significant circadian rhythms with the peak time located during the rest span (approximately =5 HALO) for both brain and liver, but during the activity span for the kidney ( approximately =21 HALO) and plasma (approximately =13 HALO). This chronobiological study showed that under physiological conditions, lipid peroxidation depends on several factors. The MDA peak/trough might be used as a tool to detect moments of high/low sensitivity of tissues to ROS attack in rodents.     

Circadian Rhythms in Toxic Effects of the Serotonin Antagonist Ondansetron in Mice

The aim of the study was to learn whether the lethal and the motor incoordination (ataxia) side effect of ondansetron (Zophren®) administration is dosing‐time dependent. Ondansetron is a serotonin 5‐HT₃ receptor antagonist used primarily to control nausea and vomiting arising from cytotoxic chemo‐ and radiotherapy. A total of 210 male Swiss mice 10 to 12 weeks of age were synchronized for 3 weeks by 12h light (rest span)/12h dark (activity span). Different doses of ondansetron were injected intraperitoneally (i.p.) at fixed times during the day to determine both the sublethal (TD₅₀) and lethal (LD₅₀) doses, which were, respectively, 3.7 ± 0.6 mg/kg and 4.6 ± 0.5 mg/kg. In the chronotoxicologic study a single dose of ondansetron (3.5 mg/kg, i.p.) was administered to different and comparable groups of animals at four different circadian stages [1, 7, 13, and 19h after light onset (HALO)]. The lethal toxicity was statistically significantly dosing time‐dependent (χ² = 21.51, p < 0.0001). Drug dosing at 1 HALO resulted in 100% survival rate whereas drug dosing at 19 HALO was only one‐half that (52%). Similarly, lowest and highest ataxia occurred when ondansetron was injected at 1 and 19 HALO, respectively (χ² = 22.24, p < 0.0001). Effects on rectal temperature were also dosing‐time related (Cosinor analysis, p < 0.0001). The characteristics of the waveform describing the temporal patterns differed between the studied variables, e.g., lethal toxicity and survival rate showing two peaks and rectal temperature showing one peak in the 24h time series waveform pattern. Cosinor analysis also revealed a statistically significant ultradian (τ ≡ 8h) rhythmic component in the considered variables. Differences in curve patterns in toxicity elicited by ondansetron on a per end point basis are hypothesized to represent the phase relations between the identified 24h and 8h periodicities.     

Pharmacological Modulation of Cisplatin Toxicity Rhythms with Buthionine Sulfoximine in Mice Bearing Pancreatic Adenocarcinoma (PO3)

In a previous report, we showed that the circadian rhythm of cisplatin (cis-diamminedichloroplatinum, CDDP) toxicity in healthy mice was modified by buthionine sulfoximine (BSO), a specific inhibitor of glutathione (GSH) synthesis. In the present study, the effects of BSO on the rhythms of CDDP toxicity and antitumor efficacy were investigated in mice bearing a transplantable pancreatic adenocarcinoma (PO3). B₆D₂F₁ mice were inoculated widi two 4 mm³ tumor fragments, one in each flank, then were synchronized with an alternation of 12h of light (L) and 12h of darkness (D) (LD 12: 12). Three weeks later, a single dose of CDDP (12 mg/kg iv) was injected at 3h, 7h, 11h, 15h, 19h, or 23h after light onset (HALO) with or without prior BSO (450 mg/kg ip 4h earlier). The antitumor activity of CDDP as assessed by tumor weight change and tumor growth delay was weak in this tumor model irrespective of prior BSO administration or CDDP dosing time. Nevertheless, toxic effects of CDDP as gauged by body weight loss or survival varied significantly according to CDDP dosing time. Body weight loss was least in mice receiving CDDP alone at the mid-to-late active span. Survival rate was 97% in mice treated with CDDP alone and 47% in those receiving prior BSO (χ² = 23.6, p <. 0001). BSO pretreatment further shifted the period of survival or body weight change from 24h to (10 + 24)h, an effect similar to that earlier reported in healthy mice. Thus, PO3 tumor at a measurable stage altered neither the circadian rhythm in CDDP toxicity nor the ultradian rhythm in the toxicity of BSO-CDDP combination. The results suggest that rhythms in target tissues for drug actions can be manipulated with biochemical modulators, thus partly escaping central clock control.     

Circadian Time‐Effect of Orally Administered Loratadine on Plasma Pharmacokinetics in Mice

Little is known about the chronopharmacokinetics of loratadine, a long‐acting tricyclic antihistamine H₁ widely used in the treatment of allergic diseases. Hence, the pharmacokinetics of loratadine and its major metabolite, desloratadine, were investigated after a 20 mg/kg dose of loratadine had been orally administered to comparable groups of mice (n=33), synchronized for three weeks to 12 h light (rest span)/12 h dark (activity span). The drug was administered at three different circadian times (1, 9, and 17 h after light onset [HALO]). Multiple blood samples were collected over 48 h, and plasma concentrations of loratadine and desloratadine were determined by high performance liquid chromatography. There were no significant differences in T_max of loratadine and desloratadine between treatment‐time different groups. However, the elimination half‐life (t1/2) of the parent compound and its metabolite was significantly longer (p<0.01) following administration at 9 HALO (t1/2 loratadine and desloratadine 5.62 and 4.08 h at 9 HALO vs. 4.29 and 2.6 h at 17 HALO vs. 3.26 and 3.27 at 1 HALO). There were relevant (p<0.05) differences in C_max between the three treated groups for loratadine and desloratadine; 133.05±3.55 and 258.07±14.45 ng/mL at 9 HALO vs. 104.5±2.61 and 188.62±7.20 ng/mL at 1 HALO vs. 94.33±20 and 187.75±10.79 ng/mL at 17 HALO. Drug dosing at 17 HALO resulted in highest loratadine and desloratadine total apparent clearance values: 61.46 and 15.97 L/h/kg, respectively, whereas loratadine and desloratadine clearances (CL) were significantly slower (p<0.05) at the other administration times (loratadine and desloratadine CL was 57.3 and 14.22 L/h/kg at 1 HALO vs. 43.79 and 12.89 L/h/kg at 9 HALO, respectively). The area under the concentration‐time curve (AUC) of loratadine and desloratadine was significantly (p<0.05) greater following drug administration at 9 HALO (456.75 and 1550.57 (ng/mL) · h, respectively); it was lowest following treatment at 17 HALO (325.39 and 1252.53 (ng/mL) · h, respectively). These pharmacokinetic data indicate that the administration time of loratadine significantly affected its pharmacokinetics: the elimination of loratadine and its major metabolite desloratadine.     

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.     

Chronopharmacologic Effects of Recombinant Human Granulocyte Colony-Stimulating Factor (rhG-CSF) on Hematopoiesis in Mice

Chronopharmacologic effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on circulating white blood cell and differential counts as well as bone marrow granulocyte-macrophage colony-forming units (CFU-GM) counts were investigated in B6D2F1 mice. The animals were synchronized with an alternation of 12 h of light (L) and 12 h of darkness (D) (LD 12:12) for 3 weeks prior to study, then received a daily subcutaneous injection of rhG-CSF (400 µg/kg/day) for 4 consecutive days at 3, 9, 15 or 21 h ours a fter l ight o nset (HALO). Samples were obtained on the fifth day at the same circadian stage as that of rhG-CSF injection. rhG-CSF significantly increased the 24-h mean of leukocyte, neutrophil, lymphocyte and CFU-GM counts. Maximum increase in leukocyte and neutrophil counts was observed when rhG-CSF was administered in the middle of the dark span, while maximum stimulatory effect on circulating lymphocytes or on CFU-GM counts was obtained with rhG-CSF administration near the middle of the light span. The results indicate that choosing the dosing time of this cytokine may selectively orient its pharmacologic action. Appropriate chronomodulated delivery schemes of rhG-CSF may further reduce hematological toxicity following chemotherapy.     

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.     

Chronopharmacologic Effects of Recombinant Human Granulocyte Colony-Stimulating Factor (rhG-CSF) on Hematopoiesis in Mice

Chronopharmacologic effects of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on circulating white blood cell and differential counts as well as bone marrow granulocyte-macrophage colony-forming units (CFU-GM) counts were investigated in B6D2F1 mice. The animals were synchronized with an alternation of 12 h of light (L) and 12 h of darkness (D) (LD 12:12) for 3 weeks prior to study, then received a daily subcutaneous injection of rhG-CSF (400 µg/kg/day) for 4 consecutive days at 3, 9, 15 or 21 h ours a fter l ight o nset (HALO). Samples were obtained on the fifth day at the same circadian stage as that of rhG-CSF injection. rhG-CSF significantly increased the 24-h mean of leukocyte, neutrophil, lymphocyte and CFU-GM counts. Maximum increase in leukocyte and neutrophil counts was observed when rhG-CSF was administered in the middle of the dark span, while maximum stimulatory effect on circulating lymphocytes or on CFU-GM counts was obtained with rhG-CSF administration near the middle of the light span. The results indicate that choosing the dosing time of this cytokine may selectively orient its pharmacologic action. Appropriate chronomodulated delivery schemes of rhG-CSF may further reduce hematological toxicity following chemotherapy.