Chronic exposure to a GSM-like signal (mobile phone) does not stimulate the development of DMBA-induced mammary tumors in rats: results of three consecutive studies.

Certain epidemiological and experimental studies raised concerns about the safety of radiofrequency (RF) electromagnetic fields because of a possible increased risk of leukemia and lymphoma. In this study, an RF field used in mobile telecommunication was tested using 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary tumors in female Sprague-Dawley rats as a model for human breast cancer. Three experiments were carried out under strictly standardized conditions and were started on the same day of three consecutive years. The field consisted of a GSM-like signal (900 MHz pulsed at 217 Hz, pulse width 577 micros) of relatively low power flux density (100 microW/cm(2) +/- 3 dB) and was applied continuously throughout each experiment to freely moving animals. The specific absorption rates averaged over the whole body were 17.5-70 mW/kg. The highest values in young animals were at or around the exposure limit permissible for the general public (i.e. 80 mW/kg). The animals were palpated weekly for the presence of mammary tumors and were killed humanely when tumors reached a diameter of 1-2 cm to allow a reliable histopathological classification and a distinction between malignant and benign subtypes. The overall results of the three studies are that there was no statistically significant effect of RF-field exposure on tumor latency and that the cumulative tumor incidence at the end of the experiment was unaffected as well. The risk ratios were 1.08 (95% CI: 0.91-1.29) and 0.96 (95% CI: 0.85-1.07) for benign and malignant tumors, respectively. These observations are in agreement with other published findings. In the first experiment, however, the median latency for the development of the first malignant tumor in each animal was statistically significantly extended for RF-field-exposed animals compared to controls (278 days compared to 145 days, P = 0.009). No such differences were detected in the two subsequent experiments. These results show that low-level RF radiation does not appear to possess carcinogenic or cancer-promoting effects on DMBA-induced mammary tumors. To explain the mechanisms underlying the different results obtained in the three experiments, a hypothesis is presented which is based upon the neuroendocrine control mechanisms involved in the promotion of DMBA-induced mammary tumors. Despite the apparent absence of stimulatory effects of low-level RF-field exposure on the development and growth of solid tumors, it will be necessary to verify these results for leukemias and lymphomas, which may have completely different biological control mechanisms.

Seasonal rhythms of 6-sulphatoxymelatonin (aMT6s) excretion in female rats are abolished by growth of malignant tumors.

The effect of development and growth of malignant tumors on pineal melatonin production was studied in two different hormone-dependent tumor systems in female rats. Urinary excretion of 6-sulphatoxymelatonin (aMT6s), the metabolic end product of melatonin, which parallels its production, was determined by radioimmunoassay at fortnightly or monthly intervals over the period of 1 year in female F344 Fischer rats bearing chemically-induced mammary carcinomas and in BDII/Han rats with spontaneous endometrial carcinomas. Untreated Fischer rats and BDII/Han rats in which tumor growth was suppressed by treatment with a progestin served as controls. Based on the cosinor analysis, animals without tumors showed significant seasonal rhythms of aMT6s excretion, with peaks in August (Fischer rats) and in May (BDII/Han rats). In contrast, such rhythms were absent in animals with developing and manifest tumors. It is concluded that animals kept under constant environmental conditions still show seasonal rhythms of pineal activity. Tumor development and growth affect pineal activity leading to disturbance of these rhythms.

Serial transplants of DMBA-induced mammary tumors in fischer rats as model system for human breast cancer: V. Myoepithelial-mesenchymal conversion during passaging as possible cause for modulation of pineal-tumor interaction.

An elevation of melatonin secretion parallel to an enhanced production of macrophage-derived biopterin was observed in female F344 Fischer rats bearing passage 2 serial transplants derived from a malignant mammary tumor induced by 7,12-dimethylbenz[a]anthracene (DMBA). As opposed to that both parameters were depressed at passage 12. These results indicate the presence of divergent immunoneuroendocrine interactions during different phases of tumor growth. Since these biochemical events must have their common origin in changes taking place within these tumor transplants the current histopathological study was initiated. The primary tumor used for serial transplantation was a moderately differentiated adenocarcinoma of the mammary gland showing cytokeratin-positive epithelial components located in the inner epithelial tubule layer. In addition, bland-looking round or elongated actin-positive myoepithelial cells were detected which apart from epithelial cells are known to constitute the main cellular components of the mammary ductal system which resemble smooth muscle cells both morphologically and functionally. The tumor of passage 1 showed glandular tubules, lined by an inner epithelial layer, and many nests of clear, bland-looking actin-positive myoepithelial cells lying around tubules as well as in the stroma between actin-negative epithelial elements. The tumor of passage 2 used for transplantation consisted of a chaotic mixture of epithelial carcinomatous cells, forming a few irregular small tubules or solid nests, and, predominantly, of elongated plump or spindle-shaped, "myoid" atypical myoepithelial cells with a strong actin-positive reaction and some of these cells showed a focal vimentin expression. The tumor was characterized as a carcinosarcoma. At passage 12 epithelial cells were not identified. The tumor displayed features of a pleomorphic sarcoma consisting mainly of giant cells with bizarre nuclei being cytokeratin- and desmin-negative, weakly vimentin-positive but strongly actin-positive. These results indicate that DMBA-induced mammary tumor cells in female F344 Fischer rats undergo dramatic morphological changes during serial transplantation characterized by a total loss of malignant epithelial (carcinomatous) cells and the emergence and subsequent predominance of malignant (sarcomatous) mesenchymal cells. It appears that these sarcomatous cells develop out of myoepithelial cells since atypical myoepithelial cells with a strong actin-positive reaction showed a focal vimentin expression at passage 2 indicating myofibroblastic differentiation as part of mesenchymal transition. The loss of epithelial cell elements as well as a parallel transition of myoepithelial to mesenchymal cell elements during passaging could lead to a lack of immunological recognition of these tumor transplants and to depression of melatonin. Possible mechanisms involved in these phenomena as well as the relevance of these findings for a better understanding of the role of melatonin in human mammary cancer are discussed.

Cloning, heterologous expression and kinetic analysis of glycerol kinase (TbGLK1) from Trypanosoma brucei.

We have cloned and sequenced the gene for the glycerol kinase of Trypanosoma brucei (TbGLK1), obtained by RT-PCR. The corresponding mRNA is 2.3 kb in size and contains an ORF encoding a protein with high homology to known glycerol kinases of other organisms. It is 512 amino acids in length with a PTS1-like targeting sequence (AKL) at its C-terminus, suggesting glycosomal compartmentalization of this enzyme. Although Northern blot analysis revealed higher mRNA levels in slender bloodstream forms than in the procyclic insect forms, specific glycerol kinase activities were found to be virtually identical in both life stages. Southern blot analysis suggested a single copy gene, but we were able to clone two alleles utmost similar to each other. Heterologous expression of the trypanosomal glycerol kinase in E. coli enabled us to perform a kinetic analysis of this enzyme. In particular, we have been able to monitor ATP production from glycerol-3-phosphate and ADP, a reaction which, although thermodynamically very unfavorable, is regarded essential for the survival of Trypanosoma brucei under anoxic conditions. Since the unique spatial separation of glycolysis in the kinetoplastida imposes important consequences for the regulation of the energy metabolism in these organisms, we discuss the observed differences between TbGLK1 and glycerol kinases from other organisms in view of its physiological relevance.

Effect of melatonin and pineal extracts on human ovarian and mammary tumor cells in a chemosensitivity assay.

Pinealectomy enhances tumor growth and metastatic spread in experimental animals. This effect is only in part due to melatonin since melatonin-free pineal extracts containing yet unidentified pineal substances have also shown tumor inhibiting activity. Despite numerous reports suggesting melatonin as a potential anti-cancer agent there have not been sufficient clinical trials to define the actual therapeutic potential of melatonin for the treatment of human cancers. To help fill this gap, we used a chemosensitivity assay designed to test the sensitivity of tumors from individual patients towards chemotherapeutic drugs for assessing the effect of melatonin and pineal extracts on primary human tumor cells. Primary cell cultures from seven ovarian and six mammary tumors were incubated with melatonin, the pineal extract YC05R (containing substances between 500 and 1000 daltons) and chemotherapeutic drugs. The pineal extract YC05R inhibited growth of all tumors in a dose-dependent manner. Physiological concentrations of melatonin (10(-8)-10(-10) M) inhibited the growth of one out of six mammary carcinomas in a dose-dependent manner. Primary cell cultures from three ovarian tumors were affected by melatonin in different ways, i.e., two were inhibited and one was slightly stimulated. There was no correlation between sensitivity towards melatonin and sex steroid receptor status, stage or grade of the tumor. It is concluded that, 1), melatonin may be an inhibitor of human mammary and ovarian carcinoma in individual cases and, 2), the pineal gland contains very active anti-tumor substances inhibiting both, the mammary and ovarian tumors, tested. These substances require chemical and biological identification.

Common myofibroblastic features of newborn rat astrocytes and cirrhotic rat liver stellate cells in early cultures and in vivo.

Double-immunolabelling techniques were employed to investigate the distribution of smooth muscle alpha-actin (actin) in glial fibrillary acidic protein (GFAP)-positive cells in rat brain during early postnatal development and maturation and in glial primary culture derived from newborn rat brain. In addition the expression of desmin was studied in the glial primary cultures as a function of the differentiation of the cells. Comparison of the cultured astroglial cells at an early age with hepatic stellate cells derived from CCl4-induced cirrhotic rat liver, revealed features of the astrocytic cytoskeleton characteristic of myofibroblastic cells, i.e., strong expression of both myofibroblastic markers, actin and desmin. In astroglial cells with an initial morphology reminiscent of fibroblasts the non-filamentous perinuclear immunoreaction of GFAP increased with time at the expense of actin and, partially, desmin. GFAP filaments were spread throughout the cytoplasm of the cells which acquired stellate morphology. The alterations in the morphology of the cells and the distribution and intensity of staining for GFAP and actin during the differentiation of astrocytes in culture were similar to those observed in astrocytes during the maturation of the brain. In astrocytes from a newborn brain as well as in cirrhotic hepatic stellate cells, the area of immunoreaction of GFAP was reduced and confined mainly to the nuclear region. In contrast, the cells expressed actin throughout the cytoplasm. These findings may hint at a similar function of these regionally specialized perivascular myofibroblastic cells in a normal brain and diseased liver and at inverse organ-specific functions which the cells fulfill under non-pathological conditions in vivo.

Serial transplants of DMBA-induced mammary tumors in Fischer rats as a model system for human breast cancer. VI. The role of different forms of tumor-associated stress for the regulation of pineal melatonin secretion.

Previous studies on human breast cancer patients showed a decline in circulating melatonin levels corresponding to primary tumor growth and an increase when relapse occurred. The aim of the current investigation was to study in an experimental model possible mechanisms involved. Inbred female F344 Fischer rats were used for serial passages derived from a chemically induced mammary adenocarcinoma. Animals with slow-growing carcinosarcomas at passage 2 showed a significant elevation of nocturnal urinary melatonin (23. 00-07.00 h; +50%, p < 0.05) and a nominal increase in plasma melatonin (+41%; 02.00-03.00 h). By contrast, these parameters were significantly depressed in animals with fast-growing sarcomas (urinary melatonin: -22%, p < 0.025; plasma melatonin: -56%, p < 0. 01). At passage 2 nocturnal pineal N-acetylserotonin (02.00-03.00 h) was significantly enhanced (+62%, p < 0.05) probably due to an increased activity of serotonin-N-acetyltransferase (SNAT, +45%), the rate-limiting step of pineal melatonin biosynthesis converting serotonin to N-acetylserotonin. The activation of SNAT may be due to a stimulation of the sympathetic nervous system (urinary noradrenaline; NA: +243%, p < 0.005) when the cellular immune system responded towards tumor growth (urinary biopterin, +214%, p < 0.005). At passage 12 SNAT and N-acetylserotonin were unaffected but a depletion of plasma tryptophan (-34%, p < 0.0001), the precursor amino acid of melatonin, was found. The marginal decline in pineal serotonin (-18%, p < 0.05) disputes that the drastic depletion in circulating melatonin (-56%, p < 0.01) can be exclusively explained by a reduced availability of tryptophan. Therefore, the involvement of an additional mechanism has to be postulated, such as a degradation of melatonin via indoleamine 2,3-dioxygenase, an extrahepatic enzyme which has been detected in tumor tissue and is related to tryptophan 2,3-dioxygenase (TDO). TDO occurs only in the liver, is highly specific for L-tryptophan and is induced by glucocorticoids which would account for the observed depletion of plasma tryptophan resulting from a tumor-associated activation of the hypothalamo-pituitary-adrenal axis (urinary corticosterone +208%, p < 0.01). These findings present first explanations for the previously observed modulation of melatonin levels in cancer patients but also illustrate the high degree of complexity of mechanisms involved in the interactions between tumor growth and the immunoneuroendocrine system.

Dynamics of glial fibrillary acidic protein distribution in cultured glomerular podocytes and mesangial cells of the rat kidney.

Glial fibrillary acidic protein (GFAP) has recently been shown to be expressed in the glomerular podocytes and mesangial cells (MC) of kidney (Buniatian et al (1998) Biol Cell 90, 53-61). The different localization of GFAP in podocytes and MC has raised the question whether this might reflect specific cellular functions. To address this question, in the present study podocytes and MC in early (2, 3 day-old), prolonged (5, 7 day-old) and late (14, 21 day-old) primary cultures from out-growths of glomerular explants were used. Double-immunolabeling studies demonstrated that podocytes transiently acquire myofibroblastic features, characterized by the expression of smooth muscle alpha-actin (SMAA) and increased perinuclear reaction of GFAP in prolonged cultures. The morphological differentiation of cobblestone-like podocytes into process-bearing cells was followed by loss of the myofibroblastic marker, SMAA, de novo expression of desmin, and distribution of GFAP, vimentin and desmin into the processes. In late culture, GFAP and SMAA were nearly absent from the podocytes which maintained the cobblestone-like morphology. By contrast, the myofibroblastic features gained by MC during prolonged culturing increased with time. A myofibroblast-like cytoskeleton of podocytes and MC similar to that of healthy astrocytes suggest an increased spectrum of functional activities of these cells during the acquisition of myofibroblastic features. In addition, the present study provides a new combination of biochemical and biological features by which podocytes and MC can be distinguished in culture.

Proteins of newly isolated mutants and the amino-terminal proline are essential for ubiquitin-proteasome-catalyzed catabolite degradation of fructose-1,6-bisphosphatase of Saccharomyces cerevisiae.

Addition of glucose to cells of the yeast Saccharomyces cerevisiae growing on a non-fermentable carbon source leads to selective and rapid degradation of fructose-1,6-bisphosphatase. This so called catabolite inactivation of the enzyme is brought about by the ubiquitin-proteasome system. To identify additional components of the catabolite inactivation machinery, we isolated three mutant strains, gid1, gid2, and gid3, defective in glucose-induced degradation of fructose-1,6-bisphospha-tase. All mutant strains show in addition a defect in catabolite inactivation of three other gluconeogenic enzymes: cytosolic malate dehydrogenase, isocitrate lyase, and phosphoenolpyruvate carboxykinase. These findings indicate a common mechanism for the inactivation of all four enzymes. The mutants were also impaired in degradation of short-lived N-end rule substrates, which are degraded via the ubiquitin-proteasome system. Site-directed mutagenesis of the amino-terminal proline residue yielded fructose-1,6-bisphosphatase forms that were no longer degraded via the ubiquitin-proteasome pathway. All amino termini other than proline made fructose-1,6-bisphosphatase inaccessible to degradation. However, the exchange of the amino-terminal proline had no effect on the phosphorylation of the mutated enzyme. Our findings suggest an essential function of the amino-terminal proline residue for the degradation process of fructose-1,6-bisphosphatase. Phosphorylation of the enzyme was not necessary for degradation to occur.

Tyrosine phosphorylation regulates cell cycle-dependent nuclear localization of Cdc48p.

Cdc48p from Saccharomyces cerevisiae and its highly conserved mammalian homologue VCP (valosin-containing protein) are ATPases with essential functions in cell division and homotypic fusion of endoplasmic reticulum vesicles. Both are mainly attached to the endoplasmic reticulum, but relocalize in a cell cycle-dependent manner: Cdc48p enters the nucleus during late G1; VCP aggregates at the centrosome during mitosis. The nuclear import signal sequence of Cdc48p was localized near the amino terminus and its function demonstrated by mutagenesis. The nuclear import is regulated by a cell cycle-dependent phosphorylation of a tyrosine residue near the carboxy terminus. Two-hybrid studies indicate that the phosphorylation results in a conformational change of the protein, exposing the nuclear import signal sequence previously masked by a stretch of acidic residues.

Nocturnal urinary 6-sulphatoxymelatonin excretion is decreased in primary breast cancer patients compared to age-matched controls and shows negative correlation with tumor-size.

In previous studies a tumor-size dependent decline of the circadian amplitude of serum melatonin was found in primary unoperated breast cancer patients, which was not due to changes of the hepatic metabolism of melatonin since its main peripheral metabolite, 6-sulphatoxymelatonin (aMT6s), showed similar serum levels. The aim of the current study was to verify these previous results by measurements of the nocturnal excretion of aMT6s in urine. The determination of aMT6s was carried out by radioimmunoassay. 17 primary unoperated breast cancer (BC) patients and 34 age-matched control patients with different types of benign gynecological diseases awaiting operation (breast diseases, n=13; ovarian diseases, n=12; and uterine diseases, n=9) were analysed. The median nocturnal urinary aMT6s excretion (22:00-6:00 hr) was significantly lower (-48%, P = 0.033) in BC patients than in controls. Controls showed a significant negative linear regression with age (r = -0.419, P = 0.014). According to multivariate linear regression analysis, BC revealed no age-dependency but a significant negative effect of increasing tumor-size on aMT6s-excretion (P = 0.036) was detected. These results confirm previous findings of a decreased pineal melatonin secretion in BC patients as well as an inverse relationship with tumor-size excluding a possible distortion due to age. The mechanisms involved are unknown but indicate that BC may lead to an impaired production of pineal melatonin. The clinical relevance of these findings from therapeutic and diagnostic point of view is discussed.

Nocturnal urinary 6-sulfatoxymelatonin and proliferating cell nuclear antigen-immunopositive tumor cells show strong positive correlations in patients with gastrointestinal and lung cancer.

The hormone melatonin plays a key role in coordinating neuroendocrine signals involved in the control of biological rhythms and also appears to be involved in the regulation of cellular proliferation. In this study on patients with gastrointestinal and lung cancer the nocturnal urinary excretion of 6-sulfatoxymelatonin (aMT6s) reflecting pineal melatonin production as well as immunohistochemically detectable proliferating cell nuclear antigen (PCNA) and melatonin were measured in corresponding tumor specimens (6 colorectal, 8 stomach, and 12 lung cancers). Strong positive correlations were detected between aMT6s and PCNA for the different types of tumors analysed (1 > or = Rs > or = 0.736, P < 0.01-0.0001). These findings provide support to the concept of an involvement of the pineal gland in malignancy and suggest that aMT6s-measurements may be considered as a non-invasive tool to estimate tumor cell proliferation. Negative correlations found between urinary aMT6s and melatonin in tumor cells (-0.735 > or = Rs > or = -0.928, P < 0.01-0.0025) could be interpreted as an effort of the pineal gland to secrete melatonin to compensate for the decrease in the number of melatonin-immunopositive cells within tumor tissue where it may possess important regulatory functions.

Gene cloning and sequencing, and enzyme purification of the malate synthase of Streptomyces arenae.

Streptomyces arenae is able to grow on acetate or ethanol as the sole carbon source. The metabolic pathway used for gluconeogenesis from C2 compounds in streptomycetes has not yet been characterized. In the course of a sequencing project we identified the gene for malate synthase (aceB), a key enzyme in the glyoxylate cycle in S. arenae. The gene was cloned and sequenced. The open reading frame of 1632 bp codes for a potential protein of 61.360 kDa. A comparison with the sequences of malate synthase from other organisms shows that the phylogenetic distance to the E. coli aceB gene is no closer than that to genes from plants or fungi. Malate synthase activity was detected in cell extracts from S. arenae. Its dependence on media conditions and on the growth phase was investigated. A purification procedure was established which allows a 188-fold enrichment of the enzyme. The molecular weight of the monomer determined by SDS PAGE confirms the weight calculated from the gene sequence. However, the holoenzyme appears to be dimeric as shown by gel filtration. All other known malate synthases from eubacteria are monomeric, while those of fungi or plants are oligomeric (di-, tri-, tetra- or octameric). The apparent Km value for glyoxylate is significantly higher than that of the malate synthases of all other species published so far. The enzyme is inactive at pH values of 7 and below; the strain cannot grow on ethanol or acetate as the sole carbon source at media pH values of 7 or below.

Pentalenolactone-insensitive glyceraldehyde-3-phosphate dehydrogenase from Streptomyces arenae is closely related to GAPDH from thermostable eubacteria and plant chloroplasts.

Streptomyces arenae produces the antibiotic pentalenolactone, a highly specific inhibitor of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). During the phase of pentalenolactone production, S. arenae expresses a pentalenolactone-insensitive GAPDH isoform; otherwise, a pentalenolactone-sensitive form is expressed. The gene of the pentalenolactone-insensitive GAPDH was cloned and sequenced. Regulatory elements typical for genes encoding antibiotic resistance and production are localized upstream and downstream of the open reading frame. No expression of pentalenolactone-insensitive GAPDH was detected in Streptomyces lividans transformed with the gene. In Escherichia coli, the gene was expressed from an induced lac promoter. Amino-terminal sequencing of the heterologously expressed GAPDH proved its identity with pentalenolactone-insensitive GAPDH from S. arenae. Sequence comparisons with GAPDH from other organisms showed a close relationship to GAPDH of plant chloroplasts, of other gram-positive bacteria, and of thermophilic gram-negative bacteria. Pentalenolactone-insensitive GAPDH differs from all closely related GAPDHs only in a few residues, none of which are directly involved in catalysis or substrate binding. The total amino acid composition is more similar to GAPDH of thermophilic species than to that of mesophilic species. The purified enzyme was moderately thermotolerant, which could be a side effect of the structural changes causing pentalenolactone-resistance.

The ATPase activity of purified CDC48p from Saccharomyces cerevisiae shows complex dependence on ATP-, ADP-, and NADH-concentrations and is completely inhibited by NEM.

The cell cycle protein CDC48p from Saccharomyces cerevisiae is a member of a protein superfamily (AAA superfamily) characterized by a common region of approximately 200 amino-acid residues including an ATP binding consensus. CDC48p purified to homogeneity showed considerable ATPase activity which could be completely abolished by preincubation with NEM in the absence of ATP. ATP protects the protein from NEM and stabilizes the otherwise labile enzyme. The ATPase activity is reversibly inhibited by NADH and shows cooperativity with its substrate ATP. The application of the in vitro ATPase activity to the identification of physiologically interacting molecules is discussed. By electron microscopy, the enzyme was shown to consist of hexameric ring structures similar to its vertebrate homologue.

Serial transplants of DMBA-induced mammary tumors in Fischer rats as model system for human breast cancer. IV. Parallel changes of biopterin and melatonin indicate interactions between the pineal gland and cellular immunity in malignancy.

Nocturnal (23.00-07.00 h) urinary melatonin and total biopterin (tBI; after acidic oxidation of reduced biopterins) were analyzed during the growth of two passages of a mammary tumor line in female F344 Fischer rats. In addition, nocturnal (02.00-03.00 h) peak concentrations of pineal melatonin in plasma were analyzed when tumors had reached comparable average tumor volumes of 25-30 cm3. Since tetrahydrobiopterin (BH4) is produced by murine macrophages in response to interferon-gamma released by activated T lymphocytes, measurements of tBI can serve to estimate the state of cellular immunity. At passage 2, a slow-growing localized carcinosarcoma, tBI showed a progressing increase during tumor growth reaching more than 200% (p < 0.05-0.005) of controls by the end of the experiment. Urinary and plasma melatonin were elevated by 30-50% (p < 0.05) and 42% respectively. At passage 12, a fast-growing metastasizing sarcoma, a depression of about 20-30% was found for tBI (p < 0.05) and urinary melatonin (p < 0.025); plasma melatonin was depleted by 70% (p < 0.005). Parallel changes of both parameters at each tumor passage indicate a close link between the pineal hormone melatonin and cellular immunity. The opposite trends observed at the two passages indicate a clear stimulation of the immune system and the pineal gland at early but inhibition at advanced stages of cancer.

Hepatic hydroxylation of melatonin in the rat is induced by phenobarbital and 7,12-dimethylbenz[a]anthracene--implications for cancer etiology.

The protective function of the pineal hormone melatonin in the etiology of cancer and carcinogenic activation is increasingly well-established. Low melatonin levels seem to parallel cancer growth. The question arises as to which factors cause the depression of melatonin levels and what the direct effects are. Melatonin is known to be metabolized in the liver by hydroxylation and subsequent conjugation yielding 6-sulfatoxymelatonin as a main product. Nevertheless, the microsomal monoxygenases catalyzing the first step have been poorly investigated. To further characterize these enzymes, typical inducers of three different sub-classes, namely phenobarbital, 7,12-dimethylbenz[a]anthracene, and 17 beta-estradiol, were administered to female Fischer rats. Circadian urinary excretion patterns of melatonin and 6-sulfatoxymelatonin were determined over a 24-hour period on the third (second) day of induction. Liver homogenates were used to monitor the in vitro conversion of melatonin or 6-hydroxymelatonin to 6-sulfatoxymelatonin. Results of both approaches showed the microsomal monoxygenases catalyzing the 6-hydroxylation of melatonin to be strongly inducible by phenobarbital and to a lesser degree by the polyaromatic hydrocarbon 7,12-dimethylbenz[a]anthracene. The dramatic depletion of circulating melatonin as a result of these induction patterns and its possible implications for oncogenesis are discussed.

Different drug metabolizing capacities in cultured periportal and pericentral hepatocytes.

Isolated and cultured periportal (PP) and pericentral (PC) hepatocytes were used for studying the acinar distribution of several phase I and phase II drug metabolizing reactions and their induction by phenobarbital (PB) and 3-methylcholanthrene (MC) or a combination thereof. Ethoxycoumarin-o-deethylase (EC 1.14.14.1) (ECOD) activity was found to predominate in PC hepatocytes even after induction with MC and PB. Metabolism of biphenyl to a monosulfated product also predominated in PC hepatocytes as did the conversion of harmine to harmine glucuronide and sulfate. In contrast, metabolism of lonazolac was not zonated. The metabolism of all three substrates declined during cultivation for 24 hr and was differentially induced (biphenyl and harmine) or not affected (lonazolac) by MC or PB. Metabolic heterogeneity was best maintained by the combination of MC and PB indicating that the zonal differences are due to a specific balance of phase I and phase II reactions. Glutathione-S-transferase (GST) activities against 1-chloro-2, 4-dinitrobenzene (CDNB) were higher in PC hepatocytes and remained so after induction with PB. In contrast, GST activities against 1,2-dichloro-4-nitrobenzene (DCNB) were almost twice as high in PP cells but equilibrated due to a spontaneous increase during cultivation, particularly in PC hepatocytes. In the presence of PB or both, MC and PB, induction of the activity against DCNB occurred exclusively in the PP hepatocytes. The distribution of the GST subunits Ya and Yb1 roughly corresponded to the pattern of GST activities against CDNB and DCNB, respectively. These results agree with earlier reports demonstrating that PP and PC hepatocytes show different patterns of phase I and phase II drug metabolizing enzymes which are maintained during short-term cultivation. In vitro induction of these activities does not result in equilibration but rather maintenance or even pronounciation of these zonal differences.

Seasonality of pineal melatonin production in the rat: possible synchronization by the geomagnetic field.

Pineal melatonin production was estimated by means of urinary 6-sulfatoxymelatonin (aMT6s) determination in two groups of female rats for 1 year each. Seasonal changes of nocturnal aMT6s excretion were found with peak levels in summer despite constant photoperiods. We hypothesize that the horizontal component H of the geomagnetic field may act as a seasonal zeitgeber because H shows a similar seasonal rhythm, and changes in the direction and intensity of H can affect pineal activity. The observed seasonal changes of pineal melatonin production stress that despite constant environmental conditions, endocrine experiments require consideration of season, neglect of which may lead to contradictory results.