Chronopharmacology of morphine in mice

Dosing-time-dependent changes in the effect and toxicity of morphine were examined in mice housed under alternating 12 h light (07:00 to 19:00 h) and dark (19:00 to 07:00 h) cycles. Morphine (0.5 mg/kg) was injected intraperitoneally (i.p.) in animals to assess its beneficial effect (i.e., protection against the kaolin-induced, bradykinin-mediated, writhing reaction) and its toxicity (i.e., alteration of the hepatic enzymes of aspartate aminotransferase [AST] alanine aminotransferase [ALT], and glutathione [GSH] in separate experiments). The magnitude of the analgesic effect of morphine depended on dosing time, with minimum effect at 02:00 h and maximum effect at 14:00 h. The serum hepatic enzyme levels of AST and ALT increased after dosing morphine (100 mg/kg) at 02:00 and 14:00 h. Time courses of these enzymes did not differ between the two trials. However, hepatic GSH, which is involved in the detoxification of chemical compounds, significantly decreased after i.p. morphine injection at 02:00 but not at 14:00 h. Overall, the results suggest that the analgesic effect of morphine is greater after dosing during the resting than during the activity phase of mice that have been induced with bradykinin-mediated pain. Drug-induced hepatic damage as inferred by GSH alteration, however, may be greater after dosing during the active phase.     

Announcements

Dosing‐time–dependent changes in the effect and toxicity of morphine were examined in mice housed under alternating 12 h light (07:00 to 19:00 h) and dark (19:00 to 07:00 h) cycles. Morphine (0.5 mg/kg) was injected intraperitoneally (i.p.) in animals to assess its beneficial effect (i.e., protection against the kaolin‐induced, bradykinin‐mediated, writhing reaction) and its toxicity (i.e., alteration of the hepatic enzymes of aspartate aminotransferase [AST] alanine aminotransferase [ALT], and glutathione [GSH] in separate experiments). The magnitude of the analgesic effect of morphine depended on dosing time, with minimum effect at 02:00 h and maximum effect at 14:00 h. The serum hepatic enzyme levels of AST and ALT increased after dosing morphine (100 mg/kg) at 02:00 and 14:00 h. Time courses of these enzymes did not differ between the two trials. However, hepatic GSH, which is involved in the detoxification of chemical compounds, significantly decreased after i.p. morphine injection at 02:00 but not at 14:00 h. Overall, the results suggest that the analgesic effect of morphine is greater after dosing during the resting than during the activity phase of mice that have been induced with bradykinin‐mediated pain. Drug‐induced hepatic damage as inferred by GSH alteration, however, may be greater after dosing during the active phase.     

Inhibiting effect of plasma from normal and tumour bearing mice on the mitotic rate of regenerating liver

Plasma from normal mice and from mice bearing the ES2 transplantable malignant tumour was injected intraperitoneally at a dose of 0.01 ml/g body weight in partially hepatectomized mice. Control animals were injected with a solution of sodium citrate in saline. The recipients were killed at the first (14:00 hours/48 h). These times are the time of day and the number of h after partial hepatectomy and second (14:00 hours/72 h) peak times after partial hepatectomy. The number of colchicine metaphases per 1000 nuclei was determined for hepatocytes and litoral cells. A different effect was obtained with plasma from tumour-bearing compared with normal mice. Plasma from both sources when injected 26 h after partial hepatectomy (16:00 hours/26 h) inhibited the mitotic activity of hepatocytes at the next peak of regenerative activity (14:00 hours/48 h). The plasma from tumour-bearing mice also inhibited the peak on the following day (14:00 hours/72 h), whereas plasma from normal mice had no inhibitory effect and, indeed, a compensatory wave was observed at this time. Furthermore, plasma from tumour-bearing mice also showed an inhibitory effect at the first peak (14:00 hours/48 h) when injected at the time of partial hepatectomy (14:00 hours/00 h) or at 22 h before partial hepatectomy (16:00 hours/-22 h) whereas the injection of plasma from normal mice at these times had no inhibitory effect. In the litoral cells the injection of plasma from tumour-bearing mice made 22 h before hepatectomy (16:00 hours/-22 h) led to a stimulation of mitotic activity which was controlled at 14:00 hours/48 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

G2 restriction point within populations of hepatocytes and tongue keratinocytes in young, growing mice

The authors studied the effect of either extracts from liver (LE) or the malignant tumour ES2 (TE) or plasma from intact mice (PI) or tumour-bearing animals (PT) on the mitotic activity of the hepatocytes and tongue keratinocytes in young, growing C3H/s male mice (28+/-1 days old). Animals standardized for periodicity analysis were injected intraperitoneally with either TE, LE, PI, PT, or saline (S) at 16:00 h with 0.01 ml of sample/g of body weight and were then killed at (time of day/h post-injection) 20:00/04, 00:00/08, and 04:00/12. Colchicine (2 microg/g) was injected 4 h before death. Samples of the liver and tongue from each animal were processed for histology and assessment of mitotic index. The results were expressed as colchicine-arrested metaphases/1000 nuclei. The TE and LE stimulated the mitotic activity of hepatocytes and tongue keratinocytes. Taking into account the time elapsed between the injections and the measurements made in these light-dark synchronized animals, we conclude that the increase in mitotic index observed in those tissues stemmed from a reinitiation of cell-cycle traverse in a subpopulation of G2-arrested, noncycling cells.     

Regulation of indoleamine 2,3-dioxygenase activity in the small intestine and the epididymis of mice

Indoleamine 2,3-dioxygenase activity was found to be ubiquitously distributed in various tissues of mice, such as brain, lung, stomach, intestine, and epididymis. The highest enzyme activity was detected in the alimentary canal and the epididymis. Developmental and daily rhythmic changes of indoleamine 2,3-dioxygenase activity and the effects of various regulatory factors were studied with the supernatant fractions derived from the small intestine and the epididymis. The enzyme activity in these two tissues was absent during the first 2 weeks (the weaning period). From the third week, there was a rapid increase in activities and a maximum was reached when the mice were 8 to 10 weeks of age (adolescence). The enzyme activity in the small intestine then gradually diminished to zero level at 30 weeks of age (prime) or later, while that in the epididymis remained at the high level throughout 69 weeks of age (senescence). The enzyme activity of the small intestine from mice fed during the hours 9:00–13:00 showed daily rhythmic changes; high in the daytime and low at night. Under night feeding (21:00–1:00), the enzyme activity was high at night and low in the daytime. The epididymal enzyme activity showed no daily fluctuations by either feeding schedule. With regard to the developmental and daily rhythmic changes, indoleamine 2,3-dioxygenase activity in the small intestine was similar to that of hepatic tryptophan 2,3-dioxygenase. However, in contrast to the hepatic tryptophan 2,3-dioxygenase activity, indoleamine 2,3-dioxygenase activity in the small intestine and the epididymis was not affected by adrenalectomy or intraperitoneal administration of adrenal steroid or tryptophan.     

Mass-spectral identification and purification of phycoerythrobilin and phycocyanobilin.

The hepatic output of triacylglycerol and cholesterol from very-low-density lipoprotein (VLD lipoprotein), and the activity of 3-hydroxy-3-methylglutaryl-coenzyme A reductase were compared in the isolated perfused rat-liver preparation and in the intact rat. The output of triacylglycerol and cholesterol from VLD lipoprotein by the perfused liver was stimulated by oleate concomitant with stimulation of hepatic microsomal hydroxymethylglutaryl-coenzyme A reductase activity. In the intact animal treated with Triton WR-1339, the magnitude of secretion of triacylglycerol and cholesterol from VLD lipoprotein coincided with the diurnal rhythm of hepatic hydroxymethylglutaryl-coenzyme A reductase activity, which was maximal at 24:00 h and minimal at 12:00 h. These observations suggest that the stimulation of the reductase and of the secretion of cholesterol from VLD lipoprotein by non-esterified fatty acids, as observed with the isolated perfused rat liver preparation in vitro, may also be an important physiological mechanism in vivo. Hepatic cholesterogenesis may be stimulated under conditions conductive to the secretion of the VLD lipoprotein, the primary transport form for triacylglycerol in the postabsorptive state.     

A blood plasma inhibitor is responsible for circadian changes in rat renal Na,K-ATPase activity

Rhythmic changes in activity following a circadian schedule have been described for several enzymes. The possibility of circadian changes in Na,K-ATPase activity was studied in homogenates of rat kidney cortex cells. Male Sprague-Dawley rats were kept on a schedule of 12h light (06:00-18:00 h) and 12 h darkness (18:00-06:00 h) for 2 weeks. At the end of the conditioning period, one rat was killed every 2 h, until completion of a 24 h cycle. Outermost kidney cortex slices were prepared, homogenized and assayed for Na,K-ATPase activity. The whole procedure was repeated six times. Na,K-ATPase activity shows an important oscillation (2 cycles/24 h). Peak activities were detected at 09:00 and 21:00 h, whereas the lowest activities were detected at 15:00 and 01:00-03:00 h. The highest activity was 40+/-3 nmoles Pi mg protein(-1)min(-1) (09:00 h), and the lowest was 79+/-3 nmoles Pi mg protein(-1)min(-1) (15:00 h). The amount of the Na+-stimulated phosphorylated intermediate is the same for the 09:00 h and 15:00 h homogenates. Preincubation of 09:00 h kidney cortex homogenates with blood plasma drawn from rats at either 03:00 h or 15:00 h, significantly inhibited their Na,K-ATPase activity. This inhibition was not seen when the preincubation was carried out with either 09:00 h or 21:00 h blood plasma. The striking oscillation (2 cycles/24 h) of the Na,K-ATPase activity of rat kidney cortex cells is ascribed to the presence of an endogenous inhibitor in blood plasma.     

Role of copper,zinc-superoxide dismutase in catalyzing nitrotyrosine formation in murine liver

The only known function of Cu,Zn-superoxide dismutase (SOD1) is to catalyze the dismutation of superoxide anion into hydrogen peroxide. Our objective was to determine if SOD1 catalyzes murine liver protein nitration induced by acetaminophen (APAP) and lipopolysaccharide (LPS). Liver and plasma samples were collected from young adult SOD1 knockout mice (SOD1(-/-)) and wild-type (WT) mice at 5 or 6 h after an ip injection of saline, APAP, or LPS. Hepatic nitrotyrosine formation was induced by APAP and LPS only in the WT mice. The diminished hepatic protein nitration in the SOD1(-/-) mice was not directly related to plasma nitrite and nitrate concentrations. Similar genotype differences were seen in liver homogenates treated with a bolus of peroxynitrite. Adding only the holo-, and not the apo-, SOD1 enzyme into the liver homogenates enhanced the reaction in an activity-dependent fashion and nearly eliminated the genotype difference at the high doses. Mass spectrometry showed four more nitrotyrosine residues in bovine serum albumin and 10 more nitrated protein candidates in the SOD1(-/-) liver homogenates by peroxynitrite with added SOD1. In conclusion, the diminished hepatic protein nitration mediated by APAP or LPS in the SOD1(-/-) mice is due to the lack of SOD1 activity per se.     

C3和C4植物叶片对光氧化响应的日变化

田间生长的C3植物花生和C4植物玉米分别于晴天上午9：00、中午12：00、下午15：00取样。中午12：00花生叶片的Fv／Fm较早上9：见下降16％,出现了光抑制现象,玉米叶片的Fv／Fm则未下降。不同时间取样的花生和玉米叶片经甲基紫精（MV） 强光的人为光氧化处理,叶绿素和类胡萝卜素出现不同程度的氧化降解,中午12：00降解幅度最大,15时降幅最小。植物叶片的抗氧化能力与其SOD活性相关,而与PEPCase的活性没有明显的相关性。光氧化处理后,花生和玉米的叶绿素荧光参数FV／Fm、qp、pSII都下降,花生在12：00的降幅最小,玉米的降幅最大。光氧化引起花生的qN和热耗散系数（KD）上升,玉米则都下降．结果显示C3植物花生和C4植物玉米对光氧化的响应可能存在不同的机制。     

小菜蛾取食后上海青的保护酶活力变化

上海青遭受小菜蛾取食为害后,0.5小时内叶片的超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD)、多酚氧化酶(PPO)和总酚含量发生变化。就一天内的变化趋势而言,受害叶片的SOD、CAT、POD、PPO活力和总酚含量总体上比正常叶片低,但在15：00时,正常叶片的SOD、CAT和POD活力最低时,受害叶片的三种酶活力却明显上升,在11：00时,正常叶片PPO活力为最低值时,受害叶片的PPO活力达最高,这可能是过度胁迫造成的,正常叶片的PPO活力与总酚量负相关,但受害叶片不呈现这种关系,此外,受害植株的未受害叶的保护酶活力也不同程度地发生变化,可见上海青对小菜蛾的取食胁迫后保护酶的反应是系统性的。     

Lipopolysaccharide-induced hepatic oxidative injury is not potentiated by knockout of GPX1 and SOD1 in mice

Knockout of copper, zinc-superoxide dismutase (SOD1) and (or) cellular glutathione peroxidase (GPX1) has been reported to have dual impacts on coping with free radical-induced oxidative injury. Because bacterial endotoxin lipopolysaccharide (LPS) triggers inflammatory responses involving the release of cytokines, nitric oxide and superoxide in targeted organs such as liver, in this study we used SOD1 knockout (SOD1−/−), GPX1 knockout (GPX1−/−), GPX1 and SOD1 double-knockout (DKO) and their wild-type (WT) mice to investigate the role of these two antioxidant enzymes in LPS-induced oxidative injury in liver. Mice of the four genotypes (2 month old) were killed at 0, 3, 6 or 12 h after an i.p. injection of saline or 5 mg LPS/kg body weight. The LPS injection caused similar increase in plasma alanine aminotransferase among the four genotypes. Hepatic total glutathione (GSH) was decreased (P < 0.05) compared with the initial values by the LPS injection at all time points in the WT mice, but only at 6 and 12 h in the other three genotypes. The GSH level in the DKO mice was higher (P < 0.05) than in the WT at 6 h. Although the LPS injection resulted in substantial increases in plasma NO in a time-dependent manner in all genotypes, the NO level in the DKO mice was lower (P < 0.05) at 3, 6 and 12 h than in the WT. The level in the GPX1−/− and SOD1−/− mice was also lower (P < 0.05) than in the WT at 3 h. The LPS-mediated hepatic protein nitration was detected in the WT and GPX1−/− mice at 3, 6 or 12 h, but not in the SOD1−/−. In conclusion, knockout of SOD1 and (or) GPX1 did not potentiate the LPS-induced liver injury, but delayed the induced hepatic GSH depletion and plasma NO production.     

Circadian rhythm of DNA synthesis and mitotic activity in tongue keratinocytes

Tongue keratinocytes have a high mitotic index (MI) with an evident circadian variation. Our study set out to compare and contrast two phases of the cell cycle: DNA synthesis (S-phase), with inmunocytochemical detection by bromodeoxyuridine (BrdU), and mitosis (M-phase), by the colchicine-arrest of metaphase method, exploring both the dorsal and ventral surfaces of the mouse tongue throughout a circadian period. Adult male mice standardized for light periodicity used for MI experiment were injected intraperitoneally with colchicine. Other animals were injected intraperitoneally with 5-BrdU for S-phase determination. Animals given both treatments were divided into six groups and killed at 4 h intervals until 20:00 h. Tongue samples were processed for histology and immuno-histochemistry. S and M indices were expressed as labelled nuclei or colchicine metaphases, respectively, per 1000 nuclei. Peak MI occurred at 12:00, with the minimum value at 20:00 on dorsal and ventral tongue surfaces. Peak S-phase was at 04:00, whereas the minimum value was at 16:00 for both surfaces. These results show that the proliferative activity of the tongue epithelium is of similar intensity and temporal distribution on both surfaces.     

Diurnal Variation of Plasmatic Melatonin,Corticosterone and Variation of General Activity in Pigeons under Light-Dark Cycle and Constant Light

This work analyzed the diurnal variation of general activity and plasmatic levels of melatonin and corticosterone in pigeons submitted to a 12:00:12:00 h light-dark cycle (lights on at 6:00 a.m.) or to constant light. In both conditions pigeons were observed in 5-min sessions at times 03:00, 06:00, 09:00, 12:00, 15:00, 18:00, 21:00 and 24:00 h during two successive days. Behavior was video taped in the home cages for posterior categorization and quantification. Radioimmunoassays were used to evaluate plasmatic levels of melatonin and corticosterone. Blood samples were obtained at the times of behavioral observation. In the light-dark condition the results showed day-night variation of general activity (p &lt; 0.001) and a robust diurnal rhythm of plasmatic melatonin (p &lt; 0.001). Both of these variations as well as the oscillatory secretion of corticosterone disappeared under constant light condition. The parallel changes in general activity and blunting of melatonin rhythm secretion in constant light condition agree with previous evidences that melatonin may regulate behavioral oscillations in the pigeon. The present data are related to the proposition that the timing system in pigeons may involve neuroendocrine relations characterized by interactions between blood born signalization by melatonin and corticosterone.     

Mice deficient in Cu,Zn-superoxide dismutase are resistant to acetaminophen toxicity

Although antioxidants are used to treat an overdose of the analgaesic/antipyretic drug APAP (acetaminophen), roles of antioxidant enzymes in APAP-induced hepatotoxicity remain controversial. Our objective was to determine impacts of knockout of SOD1 (superoxide dismutase; Cu,Zn-SOD) alone or in combination with selenium-dependent GPX1 (glutathione peroxidase-1) on APAP-induced hepatotoxicity. All SOD1-null (SOD1-/-) and SOD1- and GPX1-double-knockout mice survived an intraperitoneal injection of 600 mg of APAP per kg of body mass, whereas 75% of WT (wild-type) and GPX1-null mice died within 20 h. Survival time of SOD1-/- mice injected with 1200 mg of APAP per kg of body mass was longer than that of the WT mice (934 compared with 315 min, P<0.05). The APAP-treated SOD1-/- mice had less (P<0.05) plasma ALT (alanine aminotransferase) activity increase and attenuated (P<0.05) hepatic glutathione depletion than the WT mice. The protection conferred by SOD1 deletion was associated with a block of the APAP-mediated hepatic protein nitration and a 50% reduction (P<0.05) in activity of a key APAP metabolism enzyme CYP2E1 (cytochrome P450 2E1) in liver. The SOD1 deletion also caused moderate shifts in the APAP metabolism profiles. In conclusion, deletion of SOD1 alone or in combination with GPX1 greatly enhanced mouse resistance to APAP overdose. Our results suggest a possible pro-oxidant role for the physiological level of SOD1 activity in APAP-mediated hepatotoxicity.     

Comparative study of the activity/rest rhythms in young and old ringdove (Streptopelia risoria): correlation with serum levels of melatonin and serotonin

Aging is characterized by changes in the circadian rhythms of melatonin, serotonin, and sleep/wakefulness, alterations that affect sleep quality. The authors studied the circadian rhythms of serotonin and melatonin in young and old ringdoves (Streptopelia risoria) (2-3 and 10-12 yrs old, respectively), animals that are characterized by being monophasic and active by day, like humans. The aim was to correlate the indole rhythms with the animals' activity/rest periods. The animals were kept under a 12:12 h light/dark cycle, fed ad libitum, and housed in separate cages equipped for activity recording. Activity pulses were recorded with one actometer per animal (two perpendicular infrared transmitters) and were logged every 15 min by a computer program (DAS 16) throughout the experiment. Melatonin was measured by radioimmunoassay and serotonin by ELISA at intervals of 3 h (from 09:00 to 18:00 h) and 1 h (from 21:00 to 06:00 h), respectively. The results showed a reduction in nocturnal vs. diurnal activity of 89% and 61% in the young and old animals, respectively, with 100% considered to be the diurnal activity of each group. The amplitude of a cosine function fit to the melatonin concentrations of the old animals was half that of the young birds. The acrophase and nadir were at 02:00 and 14:00 h in the young and 01:00 and 13:00 h in the old animals, respectively. The amplitude of the corresponding cosine function fit to the serotonin concentrations in the old birds was one-third that of the young animals. The acrophase and nadir were at 15:00 and 03:00 h in the young and 16:00 and 04:00 h in the old animals, respectively. For both melatonin and serotonin, the concentrations in the young animals were significantly higher than in the old at most of the measurement times. There was a clear negative correlation between the circadian rhythms of activity and the serum melatonin levels in both young and old animals. The equivalent correlation for serotonin was positive, and stronger in the case of the young animals. The results suggest a possible relationship between the observed decline in the amplitude of the old animals' melatonin and serotonin rhythms and the lower percentage reduction in their nocturnal relative to diurnal activity pulses compared to the young animals. In conclusion, the circadian rhythms of melatonin and serotonin undergo alterations with age that could be involved in the changes in age-associated sleep.     

Effects of anthraquinone extract from Rheum officinale Bail on the physiological responses and HSP70 gene expression of Megalobrama amblycephala under Aeromonas hydrophila infection

We evaluated the effect of dietary supplementation with anthraquinone extract (from Rheum officinale Bail) on the resistance to Aeromonas hydrophila infection in Megalobrama amblycephala. The fish were randomly divided into two groups: a control group (fed a standard diet) and a treatment group (standard diet supplemented with 0.1% anthraquinone extract) and fed for 10 weeks. We then challenged the fish with A. hydrophila and recorded mortality and changes in serum cortisol, lysozyme, alkaline phosphatase (ALP), total protein, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and hepatic catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA) and the relative expression of heat shock protein 70 (HSP70) mRNA for a period of 5 d. Supplementation with 0.1% anthraquinone extract significantly increased serum lysozyme activity before infection, serum ALP activity at 24 h after infection, serum total protein concentration 12 h after infection, hepatic CAT activity 12 h after infection, hepatic SOD activity before infection, and the relative expression of hepatic HSP70 mRNA both before infection and 6 h after infection. In addition, the supplemented group had decreased levels of serum cortisol 6 h after infection, serum AST and ALT activities 12 h after infection, and hepatic MDA content 12 h after infection. Mortality was significantly lower in the treatment group (86.67%) than the control (100%). Our results suggest that ingestion of a basal diet supplemented with 0.1% anthraquinone extract from R. officinale Bail can enhance resistance against pathogenic infections in M. amblycephala.     

Orcadian Variation in Amikacin Clearance and Its Effects on Efficacy and Toxicity in Mice with and Without Immunosuppression

A once-daily dosage regimen has been recently recommended in the use of aminoglycoside antibiotics since they induce a postantibiotic effect. In choosing this regimen, one must determine the most appropriate time of day for administration of the drug. We investigated the effects of the timing of amikacin (AMK) administration on the kinetics, the efficacy against intraperitoneal infection with Pseudomonas aeruginosa, and the toxicity of AMK in mice with and without immijnosuppression. We found circadian variations in the kinetics, efficacy, and toxicity of the drug in mice. Male and female ICR mice, which were housed under a light-dark (12:12 h) cycle with free food and water intake, were injected subcutaneously with AMK sulfate 50 mg/kg body wt. There was a circadian variation in AMK clearance for both sexes with the maximum value in the dark phase and the minimum in the light phase after a single administration. When AMK 500 mg/kg/day was repeatedly administered once daily for 30 days, higher toxicity was demonstrated in mice injected with the drug at the time of day with lower AMK clearance, although no difference was demonstrated in the toxicity between the two time points with different AMK clearance when AMK 1,500 mg/kg was administered in a single dose. The ED₅₀ of AMK to cure the infected mice in the midlight phase (13:00 h) with lower clearance was significantly lower than that in the middark phase (01:00 h) with higher clearance. In contrast, the ED₅₀ in the early light phase (09:00 h) was significantly lower than that in the early dark phase (21:00 h), although AMK clearance was not different between these two different time points. In mice premedicated with cyclophosphamide to suppress immune functions, the difference in the ED₅₀ of AMK was still demonstrated between 13:00 and 01:00 h, but not between 09:00 and 21:00 h. The present study shows not only that there were circadian variations in both AMK clearance and toxicity after repeated administration, but also that there was a circadian variation in the efficacy of AMK in mice infected with P. aeruginosa. These results suggest that the timing of drug administration should be considered in pharmacotherapy with AMK and that the most appropriate time of administration in mice and nocturnal animals may be in the midlight (resting) phase. They also suggest that the ED₅₀ of AMK. against P. aeniginosa infection may be influenced not only by the circadian variation in pharmacokinetics but also by the variations in immune systems suppressed by cyclophosphamide.     

Sleepiness and Sleep in a Simulated “Six Hours On/Six Hours Off” Sea Watch System

Ships are operated around the clock using rapidly rotating shift schedules called sea watch systems. Sea watch systems may cause fatigue, in the same way as other irregular working time arrangements. The present study investigated subjective sleepiness and sleep duration in connection with a 6 h on/6 h off duty system. The study was performed in a bridge simulator, very similar to those found on ships. Twelve officers divided into two groups participated in the study that lasted 66 h. Half of the subjects started with the 06:00–12:00 h watch and the other half with the 12:00–18:00 h watch. The subjects alternated between off‐duty and on‐duty for the remainder of the experimental period. Approximately halfway through the experiment, the 12:00–18:00 h watch was divided into two 3 h watches/off‐duty periods. The effect of this was to reverse the on‐duty/off‐duty pattern between the two groups. This enabled all subjects to work the four possible watches (00:00–06:00 h, 06:00–12:00 h, 12:00–18:00 h, and 18:00–24:00 h) in an order that was essentially counterbalanced between groups. Ratings of sleepiness (Karolinska Sleepiness Scale; KSS) were obtained every 30 min during on‐duty periods and if subjects were awake during off‐duty periods. The subjectively rated duration of sleep was recorded after each off‐duty period that preceded watch periods when KSS was rated. The results showed that the average level of sleepiness was significantly higher during the 00:00–06:00 h watch compared to the 12:00–18:00 h and 18:00–24:00 h watches, but not to the 06:00–12:00 h watch. Sleepiness also progressed significantly from the start toward the end of each watch, with the exception of the 06:00‐12:00 h watch, when levels remained approximately stable. There were no differences between groups (i.e., the order between watches). Sleep duration during the 06:00–12:00 h off‐duty period (3 h 29 min) was significantly longer than during the 12:00–18:00 h period (1 h 47 min) and the 18:00–24:00 h period (2 h 7 min). Sleep during the 00:00–06:00 h period (4 h 23 min) was longer than all sleep periods except the 06:00–12:00 h period. There were no differences between groups. In spite of sufficient opportunities for sleep, sleep was on the average around 1–1 h 30 min shorter than the 7–7 h 30 min that is considered “normal” during a 24 h period. This is probably a consequence of the difficulty to sleep during daytime due to the alerting effects of the circadian rhythm. Also, sleepiness during the night and early mornings reached high levels, which may be explained by a combination of working close to or during the circadian trough of alertness and the relatively short sleep periods obtained. An initial suppression of sleepiness was observed during all watches, except for the 06:00–12:00 h watch. This suppression may be explained by the “masking effect” exerted by the relative high levels of activity required when taking over the responsibility of the ship. Toward the end of watches, the levels of sleepiness progressively increased to relatively high levels, at least during the 00:00–06:00 h watch. Presumably, initially high levels of activity are replaced by routine and even boredom.     

Orcadian Variation in Amikacin Clearance and Its Effects on Efficacy and Toxicity in Mice with and Without Immunosuppression

A once-daily dosage regimen has been recently recommended in the use of aminoglycoside antibiotics since they induce a postantibiotic effect. In choosing this regimen, one must determine the most appropriate time of day for administration of the drug. We investigated the effects of the timing of amikacin (AMK) administration on the kinetics, the efficacy against intraperitoneal infection with Pseudomonas aeruginosa, and the toxicity of AMK in mice with and without immijnosuppression. We found circadian variations in the kinetics, efficacy, and toxicity of the drug in mice. Male and female ICR mice, which were housed under a light-dark (12:12 h) cycle with free food and water intake, were injected subcutaneously with AMK sulfate 50 mg/kg body wt. There was a circadian variation in AMK clearance for both sexes with the maximum value in the dark phase and the minimum in the light phase after a single administration. When AMK 500 mg/kg/day was repeatedly administered once daily for 30 days, higher toxicity was demonstrated in mice injected with the drug at the time of day with lower AMK clearance, although no difference was demonstrated in the toxicity between the two time points with different AMK clearance when AMK 1,500 mg/kg was administered in a single dose. The ED₅₀ of AMK to cure the infected mice in the midlight phase (13:00 h) with lower clearance was significantly lower than that in the middark phase (01:00 h) with higher clearance. In contrast, the ED₅₀ in the early light phase (09:00 h) was significantly lower than that in the early dark phase (21:00 h), although AMK clearance was not different between these two different time points. In mice premedicated with cyclophosphamide to suppress immune functions, the difference in the ED₅₀ of AMK was still demonstrated between 13:00 and 01:00 h, but not between 09:00 and 21:00 h. The present study shows not only that there were circadian variations in both AMK clearance and toxicity after repeated administration, but also that there was a circadian variation in the efficacy of AMK in mice infected with P. aeruginosa. These results suggest that the timing of drug administration should be considered in pharmacotherapy with AMK and that the most appropriate time of administration in mice and nocturnal animals may be in the midlight (resting) phase. They also suggest that the ED₅₀ of AMK. against P. aeniginosa infection may be influenced not only by the circadian variation in pharmacokinetics but also by the variations in immune systems suppressed by cyclophosphamide.     

Sleepiness and sleep in a simulated "six hours on/six hours off" sea watch system

Ships are operated around the clock using rapidly rotating shift schedules called sea watch systems. Sea watch systems may cause fatigue, in the same way as other irregular working time arrangements. The present study investigated subjective sleepiness and sleep duration in connection with a 6 h on/6 h off duty system. The study was performed in a bridge simulator, very similar to those found on ships. Twelve officers divided into two groups participated in the study that lasted 66 h. Half of the subjects started with the 06:00-12:00 h watch and the other half with the 12:00-18:00 h watch. The subjects alternated between off-duty and on-duty for the remainder of the experimental period. Approximately halfway through the experiment, the 12:00-18:00 h watch was divided into two 3 h watches/off-duty periods. The effect of this was to reverse the on-duty/off-duty pattern between the two groups. This enabled all subjects to work the four possible watches (00:00-06:00 h, 06:00-12:00 h, 12:00-18:00 h, and 18:00-24:00 h) in an order that was essentially counterbalanced between groups. Ratings of sleepiness (Karolinska Sleepiness Scale; KSS) were obtained every 30 min during on-duty periods and if subjects were awake during off-duty periods. The subjectively rated duration of sleep was recorded after each off-duty period that preceded watch periods when KSS was rated. The results showed that the average level of sleepiness was significantly higher during the 00:00-06:00 h watch compared to the 12:00-18:00 h and 18:00-24:00 h watches, but not to the 06:00-12:00 h watch. Sleepiness also progressed significantly from the start toward the end of each watch, with the exception of the 06:00-12:00 h watch, when levels remained approximately stable. There were no differences between groups (i.e., the order between watches). Sleep duration during the 06:00-12:00 h off-duty period (3 h 29 min) was significantly longer than during the 12:00-18:00 h period (1 h 47 min) and the 18:00-24:00 h period (2 h 7 min). Sleep during the 00:00-06:00 h period (4 h 23 min) was longer than all sleep periods except the 06:00-12:00 h period. There were no differences between groups. In spite of sufficient opportunities for sleep, sleep was on the average around 1-1 h 30 min shorter than the 7-7 h 30 min that is considered “normal” during a 24 h period. This is probably a consequence of the difficulty to sleep during daytime due to the alerting effects of the circadian rhythm. Also, sleepiness during the night and early mornings reached high levels, which may be explained by a combination of working close to or during the circadian trough of alertness and the relatively short sleep periods obtained. An initial suppression of sleepiness was observed during all watches, except for the 06:00-12:00 h watch. This suppression may be explained by the “masking effect” exerted by the relative high levels of activity required when taking over the responsibility of the ship. Toward the end of watches, the levels of sleepiness progressively increased to relatively high levels, at least during the 00:00-06:00 h watch. Presumably, initially high levels of activity are replaced by routine and even boredom.