Relations between strain and gender dependencies of irinotecan toxicity and UGT1A1, CES2 and TOP1 expressions in mice.

Irinotecan hydrochloride (CPT-11) can display severe toxicities in individual cancer patients. CPT-11 is bio-activated through CES, detoxified through UGT1A1 and inhibits TOP1. CPT-11 toxicity and UGT1A1, CES2 and TOP1 mRNAs and UGT1A1 protein were determined in male and female C57BL/6, B6D2F1 and B6CBAF1, as potential models for tailoring CPT-11 delivery. CPT-11 was administered intravenously (40-90 mg/kg/day for 4 days at 7h after light onset). The relations between dose and lethal toxicity or body weight loss were steep and similar in C57BL/6 (lethality, p=0.001; weight loss, p=0.002) and B6D2F1 (p=0.01; p=0.03, respectively), but weak in B6CBAF1. Females displayed less toxicity than males (p<0.001). Mean mRNA expression of UGT1A1 was highest in B6CBAF1 (p=0.039) and in females (p<0.001). Both CES2 and TOP1 varied according to strain and gender (p<0.001). The three gene expression data explained the most severe toxicity of CPT-11 in male B6D2F1, but displayed inconsistent relations with toxicity in the other groups. Mean UGT1A1 protein expression was highest in males as compared to females, and so by approximately 8-fold in C57BL/6 as compared to B6D2F1 (p<0.0001). Genetic background and gender significantly altered the molecular prediction of irinotecan toxicity by UGT1A1, CES2 and TOP1 mRNA expressions.

Cross-talks between circadian timing system and cell division cycle determine cancer biology and therapeutics.

The circadian clock orchestrates cellular functions over 24 hours, including cell divisions, a process that results from the cell cycle. The circadian clock and cell cycle interact at the level of genes, proteins, and biochemical signals. The disruption or the reinforcement of the host circadian timing system, respectively, accelerates or slows down cancer growth through modifications of host and tumor circadian clocks. Thus, cancer cells not only display mutations of cell cycle genes but also exhibit severe defects in clock gene expression levels or 24-hour patterns, which can in turn favor abnormal proliferation. Most of the experimental research actively ongoing in this field has been driven by the original demonstration that cancer patients with poor circadian rhythms had poor quality of life and poor survival outcome independently of known prognostic factors. Further basic research on the gender dependencies in circadian properties is now warranted, because a large clinical trial has revealed that gender can largely affect the survival outcome of cancer patients on chronotherapeutic delivery. Mathematical models further show that the therapeutic index of chemotherapeutic drugs can be optimized through distinct delivery profiles, depending on the initial host/tumor status and variability in circadian entrainment and/or cell cycle length. Clinical trials and systems-biology approaches in cancer chronotherapeutics raise novel issues to be addressed experimentally in the field of biological clocks. The challenge ahead is to therapeutically harness the circadian timing system to concurrently improve quality of life and down-regulate malignant growth.

Preclinical relevance of dosing time for the therapeutic index of gemcitabine-cisplatin.

The relevance of gemcitabine timing for chronotherapeutic optimisation was investigated. Healthy mice received multiple doses of gemcitabine (120, 160 or 200 mg kg(-1) injection (inj)(-1)) at one of six circadian times (3, 7, 11, 15, 19 or 23 h after light onset--HALO) on days 1, 4, 7 and 10 or a single dose of gemcitabine (400 mg kg(-1)) at 11 or 23 HALO+/-cisplatin (5 mg kg(-1) at 1 min, 4 or 8 h later). Mice bearing Glasgow osteosarcoma received multiple doses of gemcitabine (200 mg kg(-1) inj(-1)) at 11 or 23 HALO+/-cisplatin (5 mg kg(-1) inj(-1) at 1 min or 4 h later) on days of 10, 13, 16 and 19 following tumour inoculation. A circadian rhythm in body weight loss was statistically validated, with 1030 HALO corresponding to the least toxic time (95% CL, 0800 to 1300). Gemcitabine dosing produced least body weight loss and least neutropenia after injection at 11 vs 23 HALO, whether the drug was given alone or with cisplatin (P=0.001). Gemcitabine-cisplatin tolerability was improved by dosing gemcitabine at 11 HALO and CDDP at 15 HALO (P<0.001). The administration of this schedule to tumour-bearing mice increased median survival three-fold as compared to treatments where both drugs were given simultaneously at 11 or 23 HALO (P=0.02). The optimal schedule would correspond to the delivery of gemcitabine upon awakening and cisplatin near mid-activity in cancer patients.

Dihydropyrimidine dehydrogenase circadian rhythm in mouse liver: comparison between enzyme activity and gene expression.

Dihydropyrimidine dehydrogenase (DPD) is the rate-limiting enzyme of 5-fluorouracil (FU) catabolism. The relevance of the measurement of DPD activity for identifying DPD-deficient patients is lessened by circadian variability in DPD activity. Our purpose was to determine whether or not DPD mRNA is sustained by a circadian rhythm. Synchronised mice (male B6D2F1) were sacrificed at 3, 7, 11, 15, 19 or 23 Hours After Light Onset (HALO; eight mice per time-point). Liver DPD activity was determined by a radio-enzymatic assay and liver DPD expression by a reverse transcriptase-polymerase chain reaction (RT-PCR) enzyme-linked immunosorbent assay (ELISA) method. Mice synchronisation was controlled by leucocyte and neutrophil counts. Individual DPD activity ranged from 555 to 1575 pmol/min/mg prot; mean DPD activity was highest at 3 HALO (mean+/-standard error of the mean (S.E.M.); 1105+/-70) and lowest at 15 HALO (889+/-71). Individual liver DPD expression varied from 761 to 3481 units (DPD/beta actin ratio); the mean was lowest at 3 HALO (1406+/-112) and highest at 15 HALO (2067+/-214). Cosinor analysis indicated that respective double amplitudes of DPD activity and expression were 21 and 30% of the 24-h mean. The acrophases for activity and expression were 6:40 and 14:10 HALO, respectively, meaning that maximum activity occurred 16 h after the maximum observed expression. These results, revealing the existence of a circadian rhythm in DPD expression, should stimulate further studies to enhance our understanding of the molecular mechanisms involved in the circadian regulation of the DPD enzyme.

Circadian optimisation of irinotecan and oxaliplatin efficacy in mice with Glasgow osteosarcoma.

The relevance of circadian rhythms in irinotecan and oxaliplatin tolerability was investigated with regard to antitumour activity. Mice bearing Glasgow osteosarcoma (GOS) received single agent irinotecan (50 or 60 mg kg(-1) per day) or oxaliplatin (4 or 5.25 mg kg(-1) per day) at one of six dosing times expressed in hours after light onset (3, 7, 11, 15, 19 or 23 hours after light onset). Irinotecan (50 mg kg(-1) per day) and oxaliplatin (4 or 5.25 mg kg(-1) per day) were given 1 min apart at 7 or 15 hours after light onset, or at their respective times of best tolerability (7 hours after light onset for irinotecan and 15 hours after light onset for oxaliplatin) or worst tolerability (15 hours after light onset for irinotecan and 7 hours after light onset for oxaliplatin). Tumour growth rate was nearly halved and per cent increase in estimated life span (% ILS) was - doubled in the mice receiving irinotecan at 7 hours after light onset as compared to 15 hours after light onset (P<0.05). Results of similar magnitude were obtained with oxaliplatin for both endpoints, yet with 7 hours after light onset corresponding to least efficacy and 15 hours after light onset to best efficacy (P<0.05). Irinotecan addition to oxaliplatin proved therapeutic benefit only if the schedule consisted of irinotecan administration at 7 hours after light onset and oxaliplatin delivery at 15 hours after light onset, i.e. when both drugs were given near their respective "best" circadian times. These would correspond to the middle of the night for irinotecan and the middle of the day for oxaliplatin in humans.

Experimental chronotherapy of mouse mammary adenocarcinoma MA13/C with docetaxel and doxorubicin as single agents and in combination.

The therapeutic index of docetaxel, doxorubicin and their combination may be improved by an adequate selection of the circadian time of administration. The present study constitutes a prerequisite to testing the clinical relevance of chronotherapy in human breast cancer. Three experiments were performed in C3H/HeN mice. Each treatment modality was administered i.v. once a week for 3 weeks at one of six circadian stages, during the light span, when the mice were resting: 3, 7, and 11 h after light onset (HALO), or during darkness, when the mice were active: 15, 19, and 23 HALO. The circadian time dependency of single agent tolerability was investigated in healthy mice using four dose levels for docetaxel (38.8, 23.3, 14, and 8.4 mg/kg/injection) and for doxorubicin (13.8, 8.3, 5 and 3 mg/kg/injection; experiment 1). The circadian time dependency of each single agent efficacy was studied in MA13/C-bearing mice, using two dose levels of docetaxel (38.8 or 23.3 mg/kg/injection) or doxorubicin (8.3 or 5 mg/kg/injection; experiment 2). The toxicity and the efficacy of the simultaneous docetaxel-doxorubicin combination were assessed as a function of dosing time in experiment 3. Two combinations were tested (A, 16.3 mg/kg/injection of docetaxel and 2.5 mg/kg/injection of doxorubicin; and B, 11.6 and 3.5 mg/kg/injection, respectively) at each of the above six circadian times. Mortality, body weight change, and tumor size were recorded for 60-70 days in each experiment. Single agent docetaxel or doxorubicin was significantly best tolerated near the middle of the rest span (7 HALO) and most toxic in the middle of the activity phase (19 HALO). Docetaxel or doxorubicin as a single drug were also most effective at 7 HALO, irrespective of dose. Treatment at 7 HALO produced highest rates of complete tumor inhibition (81% versus 11% at 3 HALO for docetaxel, p from chi2 <0.001, and 69% versus 44% at 11 HALO for doxorubicin, not significant) and highest day 60 survival rate (100% versus 28% at 3 HALO for docetaxel, p from chi2 <0.001 and 89% versus 69% at 15 HALO for doxorubicin, not significant). Docetaxel-doxorubicin combinations were most effective following dosing in the beginning of the rest span or short after the onset of the activity span, with regard to the rates of both complete tumor inhibitions (45% at 3 HALO versus 15% at 19 HALO) and day 70 survival rates (85% and 80% at 3 and 7 HALO respectively, versus 20% at 19 HALO). The efficacy of single agent docetaxel or doxorubicin and that of their combination varied largely as a function of circadian dosing time. Single agent docetaxel at 7 HALO was the best treatment option in this model with regard to both tolerability and efficacy. This timing may correspond to the middle of the night in cancer 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.

Relationship between circadian rhythm of vinorelbine toxicity and efficacy in P388-bearing mice.

The relevance of chronopharmacology for improving tolerability and antitumor efficacy of the antimitotic drug vinorelbine was investigated in female B6D2F1 mice standardized with 12 h of light and 12 h of darkness. A single i.v. vinorelbine dose (26 mg/kg) was given to 279 mice at 7, 11, 19, or 23 hours after light onset (HALO). Bone marrow necrosis and leukopenia were nearly twice as large in the mice injected at 7 HALO as compared with those treated at 19 HALO (ANOVA: p <.001 and p = 0.004, respectively). The relevance of vinorelbine dosing time for antitumor efficacy was assessed in 672 P388 leukemia-bearing mice. Vinorelbine was injected as a single dose (20, 24, 26, or 30 mg/kg) or weekly (20, 24, 26, or 28 mg/kg/injection x 3) at one of six circadian times, 4 h apart. A significant correlation between single dose and median survival time was limited to vinorelbine administration at 19 or 23 HALO. An increase in the vinorelbine weekly dose shortened median survival time in the mice treated at 7 HALO (20 mg/kg: 29 days; 24 mg/kg: 17 days; and 26 mg/kg: 6 days) but significantly improved it in those treated at 19 HALO (20 mg/kg: 28.5 days; 24 mg/kg: 32 days; and 26 mg/kg: 36 days). The study demonstrates the circadian rhythm dependence of maximum tolerated dose and the need to deliver maximum tolerated dose at the least toxic time to achieve survival improvement through chronotherapy. This may be obtained with an evening administration of vinorelbine in cancer patients.

Modulation of nonprotein sulphydryl compounds rhythm with buthionine sulphoximine: relationship with oxaliplatin toxicity in mice.

The relationship between the rhythm in tissue nonprotein sulphydryl groups (NPSH) and that in 1,2-diamine (trans-I)-cyclohexane oxalatoplatinum (1-OHP) toxicity was investigated in a total of 266 male B6D2F1 mice, using buthionine sulphoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase. Mice were synchronized with an alternation of 12 h light (L) and 12 h darkness (D; LD 12:12), and circadian time was expressed in hours after light onset (HALO). NPSH was measured in liver, jejunum and bone marrow at 0, 8 and 16 HALO. Dosing 1-OHP at these times achieved intermediate. high or low toxicity respectively. The physiological circadian rhythm in NPSH content was statistically significant in all tissues studied, with a maximum at the transition from D to L (0 HALO). BSO administration (450 mg/kg i.p., 4 h before sampling) induced a large depletion in liver and jejunum NPSH at their physiological peak (0 HALO), but exerted no significant effect at their trough (8 HALO). As a result, 24 h rhythm was suppressed in liver and jejunum, but remained similar to the physiological one in bone marrow. BSO enhanced 1-OHP-induced mortality and jejunal toxicity, but exerted no significant effect upon bone marrow toxicity. Despite these differences, 1-OHP remained least toxic at 16 HALO, near the middle of the dark span, which corresponds to maximum activity in the circadian rest/activity cycle. Our results show that mean NPSH levels in liver seem to account for the mean level of 1-OHP toxicity, while jejunal NPSH rhythm plays an important role in the intestinal toxicity rhythm of this drug.

Docetaxel chronopharmacology in mice.

Docetaxel tolerance and antitumor efficacy could be enhanced if drug administration was adapted to circadian rhythms. This hypothesis was investigated in seven experiments involving a total of 626 male B6D2F1 mice, synchronized with an alternation of 12 h of light and 12 h of darkness (12:12), after i.v. administration of docetaxel. In experiment (Exp) 1, the drug was given once a week (wk) for 6 wks (20 mg/kg/wk) or for 5 wks (30 mg/kg/wk) at one of six circadian times, during light when mice were resting [3, 7, or 11 hours after light onset (HALO)], or during darkness, when mice were active (15, 19, or 23 HALO). Endpoints were survival and body weight change. In Exp 2 and 3, docetaxel (30 mg/kg/wk) was administered twice, 1 wk apart, at one of four circadian stages (7, 11, 19, or 23 HALO). Endpoints were hematological and intestinal toxicities. In Exp 4, circadian changes in cell cycle phase distribution and BCL-2 immunofluorescence were investigated in bone marrow as possible mechanisms of docetaxel tolerability rhythm. In Exp 5 to 7, docetaxel was administered to mice bearing measurable P03 pancreatic adenocarcinoma (270-370 mg), with tumor weight and survival as endpoints. Mice from Exp 5 and 6 received a weekly schedule of docetaxel at one of six circadian stages (20 or 30 mg/kg/wk at 3, 7, 11, 15, 19, or 23 HALO). In Exp 7, docetaxel (30 mg/kg) was given every 2 days (day 1, 3, 5 schedule) at 7, 11, 19, or 23 HALO. Docetaxel dosing in the second half of darkness (19 or 23 HALO) resulted in significantly worse toxicity than its administration during the light span (3, 7, or 11 HALO). The survival rate ranged from 56.3% in the mice treated at 23 HALO to 93.8 or 87.5% in those injected at 3 or 11 HALO, respectively (Exp 1, P < 0.01). Granulocytopenia at nadir was -49 +/- 14% at 7 HALO compared with -84 +/- 3% at 19 HALO (Exp 2 and 3, P < 0.029), and severe jejunal mucosa necrosis occurred in 5 of 8 mice treated at 23 HALO as opposed to 2 of 18 receiving docetaxel at 7, 11, or 19 HALO (Exp 2 and 3, P < 0.02). The time of least docetaxel toxicity corresponded to the circadian nadir in S or G2-M phase and to the circadian maximum in BCL-2 immunofluorescence in bone marrow. Docetaxel increased the median survival of tumor-bearing mice in a dose-dependent manner (controls: 24 days; 20 mg/kg weekly, 33 days; 30 mg/kg weekly or day 1, 3, 5 schedule, 44 or 46 days, respectively; Exp 5-7). Survival curves of treated mice differed significantly according to dosing time for each dose and schedule (P from log rank <0.003 to P < 0.03). In Exp 5 and 6, the percentage of increase in life span was largest if docetaxel was administered weekly at 7 HALO (20 mg/kg, 220%; 30 mg/kg, 372%) and lowest after docetaxel dosing at 19 HALO (80% with 20 mg/kg) or at 15 HALO (78% with 30 mg/kg). In Exp 7, (day 1, 3, 5 schedule), docetaxel was most active at 11 HALO (percentage increase in life span, 390%) and least active at 23 HALO (210%). Docetaxel tolerability and antitumor efficacy were simultaneously enhanced by drug dosing in the light span, when mice were resting. Mechanisms underlying the tolerability rhythm likely involved the circadian organization of cell cycle regulation. Docetaxel therapeutic index may be improved with an administration at night in cancer patients, when fewest bone marrow cells are in S or G2-M phase.

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 with two 4 mm3 tumor fragments, one in each flank, then were synchronized with an alteration of 12 h of light (L) and 12 h of darkness (D) (LD 12:12). Three weeks later, a single dose of CDDP (12 mg/kg i.v.) was injected at 3 h, 7 h, 11 h, 15 h, 19 h, or 23 h after light onset (HALO) with or without prior BSO (450 mg/kg i.p. 4 h 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 (chi 2 = 23.6, p < .0001). BSO pretreatment further shifted the period of survival or body weight change from 24 h 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-based effects of AcSDKP, with or without rhG-CSF on hematologic toxicity of chemotherapy in mice.

The hematologic toxicity of arabinosylcytosine (Ara-C) and carboplatin (CBDCA) as well as the stimulating effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) on murine bone marrow vary according to their dosing time along the 24-h time scale. In the present study, we investigated whether the tolerability of Ara-C or CBDCA, given at their least toxic circadian time, could be improved further with AcSDKP, a negative regulator of hemopoiesis, rhG-CSF or both. A total of 228 B6D2F1 mice received once-daily injection of either Ara-C (42 mg/kg/d s.c.) for 7 d (d 0-6) at 8 hours after light onset - HALO) or CBDCA (40 mg/kg/d i.p.) for 5 d (d 2-6) at 16 HALO. AcSDKP (24 microg/d) was continuously infused for 7 d (d 0-6), using an osmotic minipump. rhG-CSF (400 microg/kg/d s.c.) was injected for 4 d (d 9-12) at 9 HALO. Subgroups of mice were sacrificed at 3 HALO on various days following treatment. AcSDKP significantly increased CFU-GM count on d 7 and leukocyte, neutrophil and monocyte counts on d 13 and d 16 compared to Ara-C alone. Also, rhG-CSF produced similar protective effects to those of AcSDKP with regard to leukocyte and CFU-GM counts. The combination of AcSDKP with rhG-CSF induced a further increase in total leukocytes and their subsets as compared to either agent alone, but did not alter the CFU-GM counts. Neither AcSDKP nor rhG-CSF nor their combination reduced CBD CA-induced hematological toxicity. In conclusion, AcSDKP or rhG-CSF administration further improved the tolerability of Ara-C beyond that already achieved with optimal circadian timing, while no such effect was observed in mice receiving CBDCA at the dose used. The results warrant further exploration of chronopharmacologic delivery schedules combining Ara-C with AcSDKP.

Pharmacologic modulation of reduced glutathione circadian rhythms with buthionine sulfoximine: relationship with cisplatin toxicity in mice.

The relationship between the rhythm in reduced glutathione (GSH) and that in cisplatin (CDDP) toxicity was investigated in a total of 560 male B6D2F1 mice, using buthionine sulfoximine (BSO). GSH was measured by high-performance liquid chromatography (HPLC) in four tissues, at each of six sampling times, 4 hr apart. A significant 24-hr rhythm was statistically validated in liver, jejunum, and colon, but not in bone marrow. Relative to liver, glutathione content was 56% in colon, 38% in bone marrow, 25% in jejunum, and negligible in kidney, where cysteine, a final product of GSH catabolism, displayed a 12-hr rhythmic variation. This rhythm may reflect that in the activity of GSH-degrading enzymes. BSO (450 mg/kg ip, 4 hr before sampling) reduced liver GSH threefold and kidney cysteine content was halved, but this pretreatment had no significant effect upon GSH content in the other organs. Furthermore, the period of the physiologic liver GSH rhythm changed from 24 hr to a composite (24 + 12 hr) period. This change in the period may result from an unmasking of the 12-hr rhythm in GSH-degrading enzyme activity by GSH synthesis blockade. Maximal values occurred in the mid-rest span and in the mid-active span after BSO administration. In the other tissues, the 24-hr period remained unchanged. BSO injection largely enhanced CDDP toxicity (as assessed by survival, leukopenia, and histologic lesions in kidney and bone marrow) and kidney mean platinum concentration. Furthermore, BSO pretreatment modified the period of CDDP toxicity rhythm: survival followed a significant 12-hr-rhythm, instead of a 24-hr rhythm. The cycling of GSH concentration results from a balance between synthesis and catabolism and likely constitutes one of the main components of the circadian rhythm in CDDP toxicity in mice.

Circadian rhythm in toxic effects of cystemustine in mice: relevance for chronomodulated delivery.

Cystemustine is a new nitrosourea with high anti-tumor activity and a short plasma half-life in mice. The influence of circadian dosing time upon its toxicities was first investigated in a total of 368 synchronized male B6D2F1 mice. Late survival rate varied from 4% in mice receiving a single dose of cystemustine (conventional lethal dose 50%) at 7 hours after light onset (HALO) up to 88% in mice treated at 15 or at 19 HALO. Target organ toxicities (bone marrow, circulating blood cells, spleen, colon and duodenum) were studied following a single slightly lower dose of cystemustine. Leukopenia was the major hematologic effect encountered. Leukocyte count nadir occurred 7 days after injection and was lowest following cystemustine at 7 HALO as compared to 13 or 19 HALO. Recovery was faster after cystemustine at 19 HALO as compared to other dosing times. Bone-marrow necrotic lesions were more pronounced 1 day after cystemustine at 7 HALO than after cystemustine at 19 HALO. Thus, a large-amplitude circadian rhythm characterized the toxicity of this nitrosourea in mice. The lowest cystemustine toxicity was found near the middle of the active span of the rest-activity circadian cycle of mice.

Modulation of cisplatin chronotoxicity related to reduced glutathione in mice.

Intracellular reduced glutathione (GSH) concentrations were measured according to the tissue sampling-time along the 24 h scale in male B6D2F1 mice. A significant circadian rhythm in GSH content was statistically validated in liver, jejunum, colon and bone-marrow (P < or = 0.02) but not in kidney. Tissue GSH concentration increased in the dark-activity span and decreased in the light-rest span of mice. The minimum and maximum of tissue GSH content corresponded respectively to the maximum and minimum of cisplatin (CDDP) toxicity. The role of GSH rhythms with regard to CDDP toxicity was investigated, using a specific inhibitor of GSH biosynthesis, buthionine sulfoximine (BSO). Its effects were assessed on both tissue GSH levels and CDDP toxicity at three circadian times. BSO resulted in a 10-fold decrease of the 24 h-mean GSH in kidney. However a moderate GSH decrease characterized liver (-23%) and jejunum (-30%). BSO pretreatment largely enhanced CDDP toxicity which varied according to a circadian rhythm. Although BSO partly and/or totally abolished the tissue GSH rhythms, it did not modify those in CDDP toxicity. We conclude that GSH have an important influence on CDDP toxicity but not in the circadian mechanism of such platinum chronotoxicity.