Natural killer cell activity: age, estrous- and circadian-stage dependence and inverse correlation with metastatic potential.

The timing within the estrous cycle of surgical removal of a transplanted murine mammary tumor profoundly influences the frequency of pulmonary metastases. We investigated the potential role of the immune response in this phenomenon by measuring splenic natural killer (NK) cell activity and interleukin-2 (IL-2) production in syngeneic tumor-free mice of two age groups at each of two circadian times and in each of four estrous stages. Estrous stage was determined by assessment of vaginal smear cellularity immediately prior to killing and spleen harvest. In a single-cell splenocyte preparation, NK cytotoxicity against a standard tumor cell target was assessed using a radiolabeled chromium release assay while IL-2 activity was determined in a bioassay utilizing the IL-2-dependent CTLL-2 cell line. Mice from the younger group were found to have eight-fold higher NK activity and 35% greater IL-2 production. After normalization of NK and IL-2 values for age, a highly statistically significant difference in NK activity was found among the four estrous and between the two circadian stages of sacrifice. NK activity was greater during the daily resting span across every estrous stage. IL-2 values were highest in diestrus and proestrus when sampled in the light span and in estrus-metestrus when sampled in the dark. The stages within the fertility cycle associated with lowest metastatic potential (proestrus/estrus) correspond precisely with those of highest splenocyte NK activity. These results indicate that an important component of the cellular immune response varies rhythmically both during the fertility and circadian cycles of the host. The rhythmic changes in NK activity may be in part responsible for the similarly rhythmic frequency of postsurgical metastatic dissemination.

Change in phase relations of circadian rhythms in cell proliferation induced by time-limited feeding in BALB/c X DBA/2F1 mice bearing a transplantable Harding-Passey tumor.

The synchronization of the circadian rhythms of [3H]thymidine uptake (as gauge of DNA synthesis and presumably of cell proliferation) in colon, thymus, and Harding-Passey melanoma were studied in 456 male BALB/c X DBA/2F1 mice under a 12-hr-light, 12-hr-dark regimen. In two groups of animals, the feeding time was restricted to 4 hr/day (either at the beginning of the light span or at the beginning of the dark span). The circadian rhythms in body temperature and [3H]thymidine uptake in the colon were determined in their timing primarily by the time of food intake. In contrast, the circadian rhythm of [3H]thymidine uptake in the thymus and in the transplanted melanoma remained synchronized with the lighting regimen, and under the conditions of this study, was not altered in its timing by the change in feeding time. It thus appears feasible to alter the phase relations between certain circadian rhythms of host and tumor. If applicable to the human situation, this observation might be of interest for the scheduling of chemo- and radiotherapy, in an attempt to obtain maximal effects upon the tumor with minimal undesired side effects upon vital functions of the host.

Reduction of cis-diamminedichloroplatinum nephrotoxicity in rats by optimal circadian drug timing.

A prominent circadian rhythm in the nephrotoxicity of a therapeutic dose of cis-diamminedichloroplatinum (cisplatin) is demonstrated in female Fischer rats. Rats were randomized to receive two doses of either cisplatin or 0.9% NaCl solution 14 days apart at the times of either high or low values in their circadian rhythm of urinary volume. Toxicity was assessed by measuring changes in body weight and changes in the 24-hr means of urinary volume, blood urea nitrogen, and urinary beta-N-acetylglucosaminidase (NAG) activity. Toxicity was least in rats which received the drug near the circadian maximum of urinary volume. Conversely, rats which received the same dose of drug near the circadian minimum of urinary volume lost more weight and exhibited a 2-fold increase in the 24-hr mean of urinary volume, a 3-fold rise in the 24-hr mean of blood urea nitrogen, and a 5-fold increase in the 24-hr mean of urinary NAG activity. A positive correlation between urinary NAG at the time of cisplatin administration and the extent of cisplatin nephrotoxicity was demonstrated (p less than 0.02). A correlation also was found between tissue NAG concentration and tissue uptake of cisplatin (p less than 0.001). A marked circadian rhythm of NAG activity in proximal tubular cells may contribute to the prominent circadian rhythm in murine renal tolerance for cisplatin.

Plasma and urinary catecholamines in salt-sensitive idiopathic hypertension.

Nineteen patients with normal renin idiopathic hypertension were arbitrarily classified as salt-sensitive or salt-resistant depending on whether their mean arterial pressure did or did not increase by 8% or more when sodium intake was increased. The responses of the two subsets and of five normal subjects to sodium intakes of 9, 109, and 249 mEq/day given for 7 days were as follows: The salt-sensitive subjects retained more sodium than normal and plasma or urinary norepinephrine did not decrease when they were given a high sodium intake; urinary dopamine was normal but did not increase normally when sodium intake was increased. The salt-resistant subjects excreted sodium normally and plasma and urinary norepinephrine was decreased by 30 and 37%, respectively, when they were given a high sodium intake; urinary dopamine was supernormal and did not increase further when sodium intake was increased. Cumulative sodium retention during the high sodium intake was directly related to the percentage of change in plasma norepinephrine in the hypertensive subjects, suggesting that renal adrenergic activity was a factor in the impaired sodium excretion in the salt-sensitive patients. Cumulative sodium retention and the percentage of change in plasma norepinephrine were inversely related to urinary dopamine in the hypertensive subjects, suggesting that increased formation of dopamine in renal and neural tissue in the salt-resistant subjects may have been responsible for the differences between the subsets in renal and adrenergic responses to a high sodium intake.(ABSTRACT TRUNCATED AT 250 WORDS)

Circadian rhythm in urinary N-acetyl-beta-glucosaminidase (NAG) of clinically healthy subjects. Timing and phase relation to other urinary circadian rhythms.

Urinary N-acetyl-beta-glucosaminidase (NAG), a lysosomal enzyme of renal tubular origin, has been shown to be a sensitive indicator of renal tubular function. This study documents a circadian rhythm in the urinary activity of NAG, statistically validated and quantified by the cosinor method, in 19 female and 15 male human subjects. The acrophase of the circadian rhythm in urinary NAG activity occurs at 09(40) with 95% confidence limits between 08(40) and 12(08) and is similar to the timing of the circadian rhythm in urinary free cortisol. The circadian acrophase of urinary NAG activity lags in timing the circadian rhythms in urine volume, Na and K excretion, and urinary free adrenalin and noradrenalin, by about five to ten hours and the circadian rhythm in creatinine excretion by about 11 hours. These functions with their characteristic phase relations are part of the internal circadian time structure of the human organism, and may provide internal phase references, independent of the "time of day." This study also documents a sex difference in mesor of the circadian rhythms in urinary NAG activity, with female subjects having a higher mesor and amplitude than the male subjects, and in the excretion of creatinine and potassium, with male subjects having a higher mesor and amplitude than the female subjects.

Splenocyte natural killer cell activity and metastatic potential are inversely dependent on estrous stage.

We have previously shown that the timing of surgical removal of an estrogen-receptor-bearing mammary adenocarcinoma within the estrous cycle of the female C3HeB/FeJ mouse profoundly influences the frequency of subsequent tumor cell metastasis. In order to investigate the role of the immune response in this phenomenon, we measured splenic natural killer (NK) cell activity and interleukin-2 (IL-2) production in 80 female cycling mice, 16 to 18 weeks old, assigned to one of four estrous stages as determined by relative quantity of vaginal cellularity; proestrus, estrus, metestrus, and diestrus. After prolonged synchronization on 12-hours-on, 12-hours-off light-dark circadian schedules, daily vaginal smears were obtained for 2 weeks to characterize estrous cycling. On the day the animals were killed, vaginal smears were performed and single-cell suspensions were prepared from the harvested spleens. Direct cytotoxicity of spleen cells against the YAC tumor target was assessed immediately in a 3 1/2 hour 51Cr release assay and expressed as NK activity in lytic units (LU 20%). IL-2 production was determined in a bioassay with the IL-2-dependent CTLL-2 cell line. Significant differences in NK activity among estrous stages mimicking the variation found in frequency of surgical cure from mammary adenocarcinoma were observed (p = 0.035; one-way analysis of variance), with the time of lowest metastatic potential corresponding precisely with the time of highest splenocyte NK activity. These both occurred during the proestrus and estrus stages, characterized by high fertility, ovulation, and peak FSH, LH, and estrogen concentrations. In addition, NK activity was found to correlate significantly with IL-2 production (r = 0.4, p less than 0.0005). These results indicate that important components of the cellular immune response to cancer vary rhythmically with hormonal changes in the host and may represent one of the factors affecting the delicate balance between host and tumor that alters the frequency of postsurgical metastatic dissemination.

Circadian pineal modulation of pituitary effect on murine corticosterone in vitro.

An old controversy is resolved as a novel effect: In a rhythmic fashion, aqueous pineal homogenate (APH) enhances, attenuates or leaves unaffected the production of corticosterone by mouse adrenals incubated with pituitary media. All glands stem from the same circadian stage in these (isophasic) studies on 72 female CD2F1 mice, standardized for two weeks in L 0600-1800 and D 1800-0600. Every 4 hours during a 24-hour span, 12 mice were killed. Pineals were removed for the preparation of APH and stored at 4 degrees C. Hypothalami, pituitaries and adrenals were removed, bisected and placed in wells containing 1 ml Krebs-Ringer buffer (K), at 4 degrees C, until incubation. At each circadian stage, bisected adrenals were incubated with 95% O2 and 5% CO2 at 37 +/- 1 degree C for 5 hours, with K only or with the addition of 0.05 IU ACTH 1-17 or APH or with isophasic pituitary or hypothalamic preincubation media with and without APH or muscle. Media were stored at -20 degrees C until corticosterone RIA. A circadian rhythm (p less than 0.05) characterized corticosterone production after stimulation by the pituitary alone or with APH. The overall modulatory effect of APH is an increased circadian amplitude of adrenal corticosterone production, in response to the isophasic pituitary.

Circannual variation of cell proliferation in lymphoid organs and bone marrow of BDF1 male mice on three lighting regimens.

BDF1 male mice, which had been raised for several generations on a lighting regimen of LD 12:12, were studied. Experiments were conducted over 24 h spans during winter, spring, summer, and fall. For 3-4 weeks prior to each study, one-third of the animals were kept on a lighting regimen of 8 h of light alternating with 16 h of darkness (LD 8:16), one-third was kept on a regimen of LD 12:12, and one-third was kept on a regimen of LD 16:8. Subgroups of mice on all three lighting regimens were killed at 4 h intervals over a 24 h span. At 20 minutes prior to sacrifice, the animals received 5 microCi of 3H-thymidine/0.2 ml/20 gm body weight intraperitoneally. The thymidine uptake in DNA (DPM[3H]/microgram DNA) was studied as an index of cell proliferation in the thymus, inguinal lymph node, spleen, femur, and a segment of the lumbar vertebral column. A circannual variation of 3H-thymidine uptake in DNA was found in all organs irrespective of the lighting regimen under which the animals were kept. The timing of the circannual variation, however, varied among the organs. In the thymus, the lowest thymidine uptake occurred during summer, with higher thymidine uptake during the other three seasons. In the inguinal lymph node, the peak in thymidine uptake was in the spring, with lower values during the other three seasons, the lowest during summer. In the spleen, the highest thymidine uptake occurred in the mice on all three lighting regimens during fall, with lower uptake during winter, spring, and summer. In the bone marrow of both the femur and the vertebral column, the thymidine uptake was high in winter and fall and low in spring and summer. Serum corticosterone measurements were available in winter, spring, and fall, and they showed statistically significant lower values in winter and fall than in spring. The conclusion was drawn that circannual rhythms of 3H-thymidine uptake in the DNA of the thymus, spleen, lymph nodes, and bone marrow are found in mice reared for generations under a LD 12:12 lighting regimen and persist if the animals are kept under a regimen of LD 8:16 or LD 16:8 for 3-4 weeks prior to sacrifice.

Circannual variation of intestinal cell proliferation in BDF1 male mice on three lighting regimens.

BDF1 male mice were studied over a 24-hr span in winter, spring, summer and fall. For three weeks prior to study, one-third of the animals were kept under a lighting regimen of 8 hr light alternating with 16 hr of darkness (LD 8:16), one-third on a lighting regimen of LD 12:12 and a remainder on a lighting regimen of LD 16:8. During each study, subgroups of animals on all three lighting regimens were killed at 4-hr intervals over a 24-hr span. Twenty minutes prior to being killed, the animals received 5 microCi of [3H]-thymidine/0.2 ml/20 gm of body weight intraperitoneally. The thymidine uptake in the DNA of the colon and of the small intestine were studied as an index of cell proliferation. A circadian rhythm in [3H]-thymidine uptake in the colon was found and validated by cosinor analysis. This rhythm was similar in acrophase and amplitude in the animals kept on LD 8:16 and LD 12:12. Also in the mice on LD 16:8, there was a statistically significant circadian rhythm of [3H]-thymidine uptake in the DNA of the colon during all four seasons. The acrophases of this rhythm, however, varied widely suggesting free running. A circadian rhythm of [3H]-thymidine uptake in small intestine was less consistent. In animals on all three lighting regimens, however, a circannual variation of [3H]-thymidine uptake in DNA in colon and small intestine was found with the highest uptake during summer. This study indicates that a lighting regimen of LD 16:8 does not reliably synchronize the circadian rhythm of [3H]-thymidine uptake in the colon. It further shows a circannual rhythm of this function in the colon and in the small intestine which persists under three lighting regimens (LD 8:16, 12:12 and 16:8) maintained for three to four weeks prior to being killed.

Circadian and circannual rhythms of hormonal variables in elderly men and women.

A group of fourteen men (73 +/- 5 yr of age), and eighteen women (77 +/- 7 yr of age) institutionalized at the Berceni Clinical Hospital, Bucharest, Romania, were studied over a 24-hr span once during each season (winter, spring, summer and fall). All subjects followed a diurnal activity pattern with rest at night and ate three meals per day with breakfast at about 0830, lunch at about 1300 and dinner at about 1830. The meals were similar, although not identical for all subjects during all seasons. On each day of sampling blood was collected at 4-hr intervals over a 24-hr span. Seventeen hormonal variables were determined by radioimmunoassay. Statistically significant circadian rhythms were detected and quantitated by population mean cosinor analysis in pooled data from all four seasons in both sexes for ACTH, aldosterone, cortisol, C-peptide, dehydroepiandrosterone-sulfate (DHEA-S), immunoreactive insulin, prolactin, 17-OH progesterone, testosterone, total T4 and TSH. In women, estradiol and progesterone also were determined and showed a circadian rhythm during all seasons. Total T3 and FSH showed circadian rhythm detection by cosinor analysis in the men only; LH showed no consistent circadian rhythm as group phenomenon in men or women. A circannual rhythm was detected using the circadian means of each subject at each season as input for the population mean cosinor in the women for ACTH, C-peptide, DHEA-S, FSH, LH, progesterone, 17-OH progesterone and TSH. In the men, a circannual rhythm was detected for ACTH, FSH, insulin, LH, testosterone and T3. There were phase differences between men and women in ACTH, FSH and LH. In those functions in which both the circadian and circannual rhythms were statistically significant, a comparison of the amplitudes showed in the women a higher circannual rather than circadian amplitude for DHEA-S. In 17-OH progesterone, TSH and C-peptide, the circadian amplitude in women was larger. In men, the circannual amplitude of T3 was larger than the circadian amplitude and in insulin the circadian amplitude was larger than the circannual amplitude. There was no statistically significant difference between the circadian and circannual amplitudes in the women in ACTH and progesterone and in the men in ACTH and testosterone.

Circadian hematologic time structure in the elderly.

Twenty-three clinically healthy, diurnally active elderly subjects, 71 +/- 5 years of age were studied over a 24-hr span (six samples). Complete blood counts and differential counts were done (Ortho ELT-8, Wright stained smears). The circadian rhythm parameters of the hematologic variables in the elderly subjects were compared with reference values obtained from a larger group of clinically healthy young adult and adult subjects studied independently. The data were analyzed by cosinor and the Bingham test. Circadian rhythms in the number of circulating formed elements in the peripheral blood persist in the aged. In comparison with the young adult, the elderly subjects show differences in the timing (phase advance) of the circadian rhythms in circulating neutrophil leukocytes and lymphocytes, a decrease in the circadian amplitude of circulating platelets, a decrease in circadian rhythm adjusted mean (mesor) in the red cell count, and in the neutrophil band forms.

Circadian rhythm in mammary cytoplasmic estrogen receptor content of Balb/C female mice with and without pituitary isografts.

Cytoplasmic estrogen receptors were determined by the dextran-coated charcoal method in inguinal breast tissue of three groups of Balb/C female mice 6-8 weeks following subcutaneous implantation into the intact animals of three pituitary glands and three pieces of skeletal muscle (group I), three pituitary glands and three segments of hypothalamic tissue (group II), or three pieces of skeletal muscle (group III) obtained from animals of the same inbred strain as control. A circadian rhythm in estrogen receptor content was statistically quantified by cosinor analysis in the muscle implanted control and the pituitary and hypothalamic implant groups. In the pituitary and muscle implant group the circadian rhythm is of borderline significance with a P-value between 0.05 and 0.10. The timing (acrophase) and extent of change (amplitude) are similar in all three treatment groups. The average receptor content (MESOR) in the two pituitary-implanted groups, which in previous studies were shown to have an increased breast cancer incidence is about twice that of the control group. The reduction in the pituitary induced breast cancer rate by hypothalamic tissue addition to a cancer incidence between the animals with pituitary and muscle isograft and the mice carrying no pituitary at all has also been shown previously in this strain of mice and is not reflected in receptor content.

Cisplatin chronotherapy and disulfiram rescue reduce toxicity without interfering with anticancer activity: animal findings and preliminary clinical experiences.

Cisplatin-induced mortality and nephrotoxicity are each predictably worse when the drug is given at certain points within the circadian schedule. Oral disulfiram protects rats from toxic effects at some circadian stages but not others. This manuever does not diminish the anticancer activity of cisplatin in these rats. Human beings given 2 g of oral disulfiram and high doses of cisplatin at the circadian stage associated with least cisplatin nephrotoxicity (prospectively determined potassium excretion acrophase) suffer little or no kidney damage. Disulfiram administration apparently does not interfere with the antineoplastic activity of cisplatin in humans. This is the first demonstration of the feasibility of assignment of treatment time according to a measure of the patient's 'internal clock' as assessed by pretreatment marker rhythmometry. It also establishes the feasibility of giving disulfiram to human beings.

Circadian characteristics of urinary epinephrine and norepinephrine from healthy young women in Japan and U.S.A.

Clinically healthy diurnally active young adult women were studied during the same season (March) at the Universities of Kyushu (Fukuoka City, Japan) and of Minnesota (Minneapolis, U.S.A.), under comparable conditions, except that the habitual diets were not changed. The subjects (20 Japanese and 16 Americans of mixed Caucasian background) were studied over a single 24-hr span. Urine was collected at 4-hr intervals. A circadian rhythm in total urinary norepinephrine excretion showed similar characteristics in Japanese and Americans. In epinephrine excretion, the Japanese women showed a statistically significantly higher amplitude with higher peak values, but no statistically significant difference in the rhythm-adjusted mean. This intergroup difference is strictly time dependent; it does not come to the fore in urine samples covering the nocturnal rest span of the subjects.

Cancer marker assessment: case report on salivary and urinary CEA.

A circadian rhythm is demonstrated for salivary CEA in a clinically healthy man who collected unstimulated saliva samples around the clock for 4 days. Its acrophase occurs around 07:00, slightly later than for patients with colon cancer. A circadian rhythm of borderline statistical significance is found for the urinary excretion rate of CEA determined during the same span by this patient. It has an acrophase occurring around 15:00, differing from that of salivary CEA. Although CEA may have only limited value to assess tumor burden, even when determined in blood, rhythm characteristics of tumor markers such as CEA await applications for guiding treatment timing and for detecting earliest chronome alterations not only in the case of an overt cancer but as a feature of predisease and/or disease risk elevation.

Screening biological samples for underivatized drugs using a splitless injection technique on fused silica capillary column gas chromatography.

A rapid screening technique is presented for the detection of underivatized drugs using a multi-level temperature program. This allowed for the identification of major clinically significant drugs extracted from serum, urine, and other body fluids. Acidic or basic extractions are injected into a gas chromatograph equipped with a flame ionization detector and a single wide-bore, thick film capillary using the splitless mode of injection. Identification is by retention time relative to that of a common internal standard. Deactivation of the fused silica liner is necessary to consistently detect low concentrations of specific drugs.

Chronobiology in laboratory medicine.

A critical amount of information has accumulated over the last decades to allow the application of chronobiology to clinical and laboratory medicine. The tasks faced in laboratory medicine include the quantitative measurement of the multifrequency human time structure in health and disease. For this purpose, it is essential to choose an adequate sample size in order to obtain meaningful results and quantitative endpoints which can be interpreted by inferential statistical techniques. No statistical technique is applicable for all purposes and it is essential that the assumptions underlying each technique and its limitation are well known to the investigator. The multifrequency nature of the human time structure has to be kept in mind in order to avoid erroneous results. Time qualified reference ranges have to be established for high amplitude rhythms. Circadian and/or circannual rhythm alterations have been described as group phenomenon in subjects with epidemiologically determined risk states for common diseases, but will require much further studies for the application to individual subjects. Rhythm parameters are new endpoints in the evaluation of the human time structure in health. Alterations of these parameters may occur as cause or as consequence of disease. Recognition of rhythm abnormalities in disease are critical for a meaningful application of chronopharmacology. Time dependent changes in pharmacokinetics and pharmacodynamics have to be taken into account in the interpretation of drug level determinations. A considerable degree of individuality of timing has been documented in some frequencies. This individuality and the rhythm abnormalities found in disease require the study of reference or marker rhythms. If the complexity of the human time structure is clearly understood and its study pursued in a critical manner with quantitative endpoints, chronobiology opens a new dimension in laboratory and clinical medicine.