The Candida albicans PMM1 gene encoding phosphomannomutase complements a Saccharomyces cerevisiae sec 53-6 mutation. We have constructed an ordered-array genomic DNA library of the pathogenic dimorphic fungus Candida albicans which facilitates the rapid cloning of C. albicans genes by hybridisation. Using the Saccharomyces cerevisiae SEC53 gene encoding phosphomannomutase as a hybridisation probe we have cloned the C. albicans homologue, PMM1, and determined its sequence. This gene shows high similarity, both at the nucleotide (76.2%) and amino-acid (77.7%) level, to the S. cerevisiae SEC53 gene. We have used the C. albicans PMM1 gene, in single copy, to transform temperature-sensitive S. cerevisiae sec53-6 mutant cells, which are defective in PMM activity at 37 degrees C, to growth at 37 degrees C. The C. albicans PMM1 gene is thus the structural and functional equivalent of the SEC53 gene.


Genome organization of mitochondrial DNA from the non-saccharomycete yeast Arxula adeninivorans LS3. Mitochondrial (mt) DNA of the asexual ascomycetous yeast Arxula adeninivorans LS3 was isolated and characterized. The mtDNA has a GC content of 30.3 mol%. It is circular and its size, as estimated by restriction analysis performed with nine endonucleases, was 35.5 kbp. Using mt gene-probes from Saccharomyces cerevisiae six structural genes (cob, cox1, cox2, oli1, oli2, and 21S rRNA) were located on the mitochondrial genome of A. adeninivorans. The comparison between the mt genomes of A. adeninivorans and other yeasts showed differences in genome organization.


[Pathological and immunohistochemical study on 63 cases of brain metastases from the lung]. Pathologic analysis of 63 cases of brain metastases from lung was reported. The specimens of all cases were labelled by 4 antibodies (HLC3-AB, CEA, Keratin, GFAP), by means of ABC method. According to our data, brain metastases from lung accounted for 50.4% of whole brain metastases. Lobus frontalis was the most common intracranial metastatic site of lung cancer. Four histopathological types were classified: adenocarcinoma (55.6%), undifferentiated carcinoma (17.4%), adenosquamous carcinoma (14.3%) and squamous cell carcinoma (12.7%). The immunohistochemical results showed that HLC3-AB is a helpful marker in identifying brain metastases from lung cancer. GFAP showed negative in malignant cells but positive in stroma. This result suggested that the stroma of brain metastases contains ingredients originating from astro-gliofibre.


[Diagnostic study of pulmonary mass under ultrasonography guided aspiration biopsy by four types of needle]. One hundred and thirty patients with pulmonary mass were studied under ultrasonography guided aspiration biopsy by four types of needle. The cytological positive cases were 57.1% in fine needle (22G), 79.6% in large needle (18G), 90.9% in canaliculated needle (18G) and 80.9% in cutting needle (14G), and the pathological positive cases were 0, 64.4%, 86.3% and 92.8% respectively. These data showed that the positive rate of fine needle puncture was the lowest one. Based on the pathological finding, canaliculated and cutting needle seem to be better than large one and it is the best choice to perform the pulmonary aspiration biopsy.


[Measurement of right and left heart function of COPD and cor pulmonale by radionuclide ventriculography]. Non-invasive measurements of right and left ventricular ejection fraction (RVEF, LVEF) by multiple-gated equilibrium radionuclide ventriculography were performed in 19 control subjects, 55 patients with COPD and cor pulmonale, simultaneous right heart catheterizations were performed in 10 patients with cor pulmonale to determine the mean pulmonary artery pressure (mPAP), and then, the acute hemodynamic and functional effects of nifedipine were evaluated. The mean RVEFs are different significantly among the various groups. With the development of the diseases, the RVEFs reduce gradually. The mean LVEF reduces significantly in cor pulmonale patients with heart failure. The RVEF correlated negatively to mPAP (r = -0.7047, P < 0.01). After nifedipine (20mg), the RVEF and mPAP do not change significantly (P > 0.05), but the artery blood pressure reduces significantly. We conclude that the equilibrium radionuclide ventriculography may be a useful and accurate method in diagnosing early cor pulmonale and cor pulmonale with right heart failure, and nifedipine may not be a good vasodilator for pulmonary hypertension.


[Effects of diprophylline on pulmonary hypertension in patients with chronic pulmonary disease]. The present study was undertaken to evaluate the short-term effects of diprophylline on hemodynamic and gases exchange in 10 patients in stable condition with chronic pulmonary disease. Diprophylline decreased the pulmonary vascular resistance by markedly reducing the mean pulmonary arterial pressure but didn't change cardiac output significantly. Diprophylline also caused a significant decrease in arterial carbon dioxide tension, but there was no significant difference between the level of PaO2, SaO2 and A-aDo2 before and after the administration of diprophylline.


[Examination of maximum transdiaphragmatic pressure, diaphragm electromyography in patients with obstructive emphysema]. This paper reported the methods and results of maximum transdiaphragmatic pressure (Pdi max) and diaphragm electromyography (EMGdi) in 16 patients with obstructive emphysema. A correlation analysis showed that Pdimax is negatively related to PaCO2, FRC%, and positively to VC, IC, FVC and MVV closely. Diaphragmatic fatigue was induced by inspiratory resistive loaded breathing with the parameters of 60% Pdimax and 5 minutes lasting. The influential effects and clinical value of diaphragm examination were discussed.


[Studies of bronchoalveolar lavage cells during the airborne Klebsiella pneumoniae infection in immunocompromised mice]. We compared immunocompromised mice with normal mice during the airborne Klebsiella pneumoniae (KP) infection, to study the course and developing mechanisms of KP pneumonia. There are significant difference in the number and peaking time of PMN, the number of lymphocytes and plasma cells in the bronchoalveolar lavage fluids of different mice groups. These results indicate that the interference of immune specific and nonspecific host responses is an important variable in antibiotic efficacy and the existence of an immunomodulating cytokine network was suggested.


[The clinical significance of red cell immune adherence function in patients with pulmonary tuberculosis and silicotuberculosis]. In our study, we observed that (1) High RCIA were discovered in these two groups. (2) In the group of pulmonary tuberculosis, the C3b receptor activities remained no change after antituberculous therapy, but the amounts of immune complexes bearing on RBC decreased. In the patients with the high RCIA, CIC was discharged rapidly and the lesions of tissue produced by CIC were prevented. In the patients with silicotuberculosis, both silica and tuberculous bacilli may act as immune adjuvant and enhance the hypersensitivity. So the C3b receptor activities were higher in silicotuberculosis patients.


[The surgical emergent therapy for massive hemoptysis in 52 cases]. The result of surgical emergent operation in 52 cases with massive hemoptysis failed by medical therapy was reported. Hemoptysis ceased in 51 of the 52 cases. One patient died after operation, giving a mortality rate of 1.9%. Other complications were occurred in two patients, but none was bronchial fistula. The complication rate was 3.8%. The surgical indication of this series would include: (1) The amount of hemoptysis more than 600 ml per 24 h, failed by medical treatment. (2) Massive hemoptysis repeated or a history of suffocation. (3) Irreversible lesion in the lung with the bleeding site identified accurately. (4) The general condition and vital organs of the patient would permit surgical therapy. Various types of pulmonary resection with successful results should be selected.


Chronolab: an interactive software package for chronobiologic time series analysis written for the Macintosh computer. Methods based on periodic regression have been designed for the detection of periodic components in short, noisy, and nonequidistant time series (as they are usually present in medicine and biology). The procedure consists of fitting a set of (cosine) curves to the data, with the analyst choosing the domain of trial periods to be analyzed and the distance between consecutive trial periods. We here describe an interactive program for least-squares rhythmometry written in C language for the Macintosh computer. For any given number of time series to be analyzed at once, the program is able to perform two different kinds of analyses: (a) linear in time, for the sequential fit of trial periods; and (b) linear in frequency, for the sequential fit of harmonic components from an initial fundamental period. For each series and for each trial period fitted to the data, the program gives the following information: fitted period; percent rhythm; p value from testing the assumption of zero amplitude; rhythm-adjusted mean or mesor, amplitude, and acrophase, each with corresponding standard errors and 95% confidence intervals when the component is statistically significant; and (when required by the analyst) p values from tests of sinusoidality, normality of residuals, and homogeneity of variance. Additionally, the program provides a summary report for each time series analyzed, including descriptive statistics such as the number of data analyzed for that series, minimum, maximum, arithmetic mean, standard deviation, standard error, 90% range, and 50% range. The analyst is also able to transform the data before doing any rhythmometric analysis. Transformations already integrated in the program include square root, logarithm, inverse, data as percentage of mean, data as percentage of mesor, and elimination of values outside +/- 3 SD from the mean. When several periods are suspected to be statistically significant, a multiple-component analysis can be also used by the concomitant least-squares fit of several harmonics. The program allows the simultaneous analysis of several periods in several variables from several individuals, with limitations depending solely on internal memory availability and speed requirements from the user. When series from different subjects or different variables in the same subject are available for analysis, a parameter test also included in the program can be used for comparison of rhythm characteristics at any given period. All information required in a single analysis is given by the analyst in the form of self-explanatory commands grouped in different "menus."(ABSTRACT TRUNCATED AT 400 WORDS)


Pinealectomy and LL abolished circadian perching rhythms but did not alter circannual reproductive or fattening rhythms in finches. In the subtropical finch, spotted munia (Lonchura punctulata), circanual rhythms (of gonads, fattening, feeding) have been demonstrated in an information-free environment of continuous illumination (LL), rendering it an ideal model for research on the physiology of the circannual clock. In an attempt to understand the involvement, if any, of the circadian system in the genesis of circannual rhythms, we studied the effect of pinealectomy (LL 15 lux) and strong continuous illumination (LL 300 lux), both known to abolish circadian rhythms, on the circadian perch-hopping rhythm and on the circannual rhythm of reproduction and fattening in the same birds. While both pinealectomy and LL 300 lux treatments abolished the circadian rhythm of motor activity, they had no effect on the circannual rhythms of gonadal size and fattening. If the endogenous circadian rhythm in perch-hopping can be taken to reflect the circadian clock mechanism associated with gonadal functioning, present results suggest that circannual rhythm of reproduction in spotted munia is independent of circadian events.


Time and sex in the male mouse: temporal regulation of infanticide and parental behavior. Infanticide is a violent but successful reproductive strategy found in many mammals, particularly rodents. In male house mice (Mus domesticus and M. musculus), the act of ejaculation provides a reliable neural signal for timing the birth of their offspring. However, a unique chronobiological aspect of this phenomenon is the extraordinary temporal latency that can occur between the stimulus (coital ejaculation) and its adaptive neural response (male mice cease killing pups and behave parentally toward them instead). Specifically, the inhibition of infanticide is often time delayed for many days after a male ejaculates, but virtually always occurs before or around the time his own sired offspring would be born 18-20 days later. Furthermore, infanticide spontaneously reemerges 50-60 days after mating. In CF-1 stock male mice this entire behavioral sequence is synchronized with the female's reproductive cycle, and occurs even in the total absence of social cues or changes in pituitary or gonadal hormones after mating. When entrained and mated at 22 h (light:dark 11:11) or 27 h (light:dark 13.5:13.5) T-cycles, photoperiodic cues appeared to synchronize this dramatic shift in behavior, because a sudden transition from pup killing to parenting was matched with the number of light/dark cycles experienced after ejaculation rather than the amount of real time experienced, suggesting a circadian timing link. Housing in constant light accelerated the postmating transition to parenting, whereas constant dark significantly delayed the transition to parenting, but still occurred by 3 weeks after mating. Most males tend to oscillate between infanticide and parental behavior for several days before locking in to constant parenting, regardless of lighting conditions. Variation in the time delay between ejaculation and the inhibition of infanticide was consistent within young individuals (< 10 months of age), but in older males (> 18 months of age) the time interval between ejaculation and parenting was significantly prolonged and attenuated. Another unique aspect of this phenomenon is that variation among individuals in their timing and response to light cues is correlated with phenotypic variation in sex steroid exposure during late fetal development. So far, no simple physiological explanation can account for the neural mechanism triggered by ejaculation that coordinates these time-delayed behavioral changes toward pups.


Chronopharmacology of methotrexate pharmacokinetics in childhood leukemia. Survival has been shown to improve when maintenance therapy for acute lymphocytic leukemia in children is given at night rather during the day. We examined the possibility that diurnal variation in methotrexate pharmacokinetics may contribute to this improvement. In a crossover study, we determined the pharmacokinetics of intravenous methotrexate at 10:00 and 21:00 h in six children with standard or high-risk leukemia. During the study, children refrained from concomitant drugs (6-mercaptopurine and trimethoprim sulfamethoxazole). There was a significant fall in methotrexate plasma clearance at night (from 5.6 +/- 3 ml/min/kg to 4.7 +/- 2.3 ml/min/kg p < 0.05). Renal clearance of methotrexate tended to decrease at night and unbound renal clearance decreased significantly (from 17.5 +/- 1.7 ml/min/kg to 8.5 +/- 3.6 ml/min/kg p < 0.05). Creatinine clearance did not exhibit diurnal variation, when comparing two 12-h collections, but there was a significant decrease in the nonglomerular clearance of methotrexate (from 14.8 +/- 5.2 to 6 +/- 4 ml/min/kg). Because it is a weak organic acid, the tubular secretion of methotrexate depends on urinary pH. At night urinary pH is more acidic. This may result in more reabsorption and hence reduced renal clearance.


Diurnal variation in mental activities of French pupils and influence of test protocol. The aim of the study was to determine the influence of the test protocol (individual vs. group) on the diurnal variations in mental activity of 18 11-year-old pupils. Number cancellation, a spatial orientation test, and mathematical problems were tested four times on three successive Fridays at 08:45, 11:15, 13:45 and 16:45 h. On the first and third Fridays the tests were performed on the group of subjects, whereas individual subjects were tested on the second Friday. Results indicated a possible influence of the test protocol (individual vs. group) on intellectual rhythmicity. Only in the group protocol did we find the psychological profile which is normally observed, namely, a performance increase during the morning, a postlunch fall, and then an increase in the afternoon. We suggest the existence of some group effect in these performance tests.


Circadian rhythm in plasma noradrenaline of healthy sleep-deprived subjects. Under normal sleep-wake conditions, noradrenaline (NA) secretions in supine subjects exhibit a weak circadian variation with a peak that occurs around noon; the sleep span is characterized by reduced NA secretion. Some investigators have reported that the circadian NA rhythm is completely obliterated during sleep deprivation. In our laboratory, plasma NA was assayed every hour for 24 h in nine healthy men 20-23 years of age. All men were deprived of sleep and were required to eat and walk around every hour to prevent sleep. However, subjects remained supine for 20 min before blood samples were collected to eliminate the effect of activity. Persistence of a slight decrease in the night concentration in several subjects, despite sleep deprivation, suggests that NA secretion may be influenced by a biological clock whose activity becomes visible when the influence of posture is removed.


Circadian phase-dependent protein and tissular binding of three local anesthetics (bupivacaine, etidocaine, and mepivacaine) in mice: a possible mechanism of their chronotoxicokinetics? The aim of this study was to investigate the possible influence of the time of administration on bupivacaine (B), etidocaine (E), and mepivacaine (M) protein and tissue (brain and heart) binding. For each anesthetic agent, a single dose of B (20 mg/kg), E (40 mg/kg), or M (60 mg/kg) was administered intraperitoneally at 10:00, 16:00, 22:00, and 04:00 h. Blood and tissue samples were collected 15 min after drug administration. This study documents significant circadian variations in protein and tissue binding of the three local anesthetic agents. We did not demonstrate a temporal relationship between the respective free and tissue levels. Thus, the temporal variations of free plasma, brain, and heart levels do not seem to be involved in the temporal changes of induced mortality.


[A morphometric grading study on the nucleus shape and DNA-content of transitional cell carcinoma of urinary bladder]. Totally 30 cases of TCC of the urinary bladder were analyzed with IBAS-2000 image analysis system. The nuclear area, perimeter, ellipse factor, etc and nucleus DNA content were measured. The morphometric grading was performed with five correlative factors for classification. They are nucleus perimeter, nucleus ellipse factor, DNA SDE, probability of the cells in C6 zone DNA and probability of the cells in C8 zone DNA. The coincident rate between morphometric grading and WHO grading was 88.9% in G1, 66.7% in G2, and 83.3% in G3. The results indicate that morphometric analysis is a valuable tool in the objective grading of bladder carcinoma.


[Ultrastructural and morphometric studies on lesions in experimental membranous glomerulonephritis (MGN) of rabbits]. Lesions of experimental MGN in rabbits were studied by transmission and scanning electron microscopy, and the size of epithelial deposits as well as the thickness of GBM were measured by using stereological quantitative method in image analysis system. Results showed that changes of the morphological parameters could be measured and analysed objectively with accuracy. Morphometric study is beneficial to investigate the relationship between morphological lesions and functional changes, and it can also provide quantitative data of the morphological change in evaluating the curative effect of drugs for glomerulonephritis.


[The effect of transfused marrow monocytes on Masugi nephritis in rabbits with radiation injury]. Sheep anti-rabbit glomerular basement membrane serum (NTS) was given to 2 groups of rabbits with a dosage of 0.8ml/kg for establishing model of crescentic glomerulonephritis. Seven days after the inoculation each rabbit received 800 rad whole body gamma-radiation in order to deplete the circulating leukocytes. Another 2 days later, all of the rabbits in group 1 received 10(8) cultivated rabbit marrow monocytes, while in group 2 nothing was given. In group 1, predominant infiltration of macrophages and cellularly crescents were obtained in the glomeruli 7 days after the administration of the cultivated cells. The Bowman's capsules were broken with crescents formation. In rabbits of group 2, there were swellings of some epithelia and endothelial cells and crescents were scanty. The result proves that the macrophages are the main factors during the formation of crescent.


[HLA-DR expression on hepatic cells in hepatitis B]. HLA-DR immunohistochemistry of 110 liver biopsies including 85 hepatitis (in 7 of them immunoelectron microscopy was done), 15 other liver diseases and 10 normal livers were studied. In the hepatitis group, Kupffer cells, sinusoidal walls, inflammatory cells and necrotic areas were strongly positive for HLA-DR; whereas in other liver diseases, they were only weakly positive. In hepatitis, CAH showed the strongest staining and a "reticular pattern". Expression of HLA-DR on hepatocyte membrane was only found in a few hepatocytes. In 5 cases of double labelling, no significant relation between HLA-DR and HBcAg was found. Immune EM identified both membrane and cytoplasm of Kupffer cells to be HLA-DR positive. Hepatocytes in focal necrosis and submassive necrosis were positive, especially in the endoplasmic reticulum. Between some hepatocytes, consecutive positive lines were found indicating that the membrane of hepatocyte could express HLA-DR.


[The effect of fluorine on the developing human brain]. Fifteen therapeutically aborted fetuses at the 5th-8th gestation month from the endemic fluorosis area were compared with those from the non-endemic area. Stereological study of the brains showed that the numerical density of volume of the neurons and the undifferentiated neuroblasts as well as the nucleus-cytoplasm ratio of the neurons were increased. The mean volume of the neurons was reduced. The numerical density of volume, the volume density and the surface density of the mitochondria were significantly reduced. The results showed that chronic fluorosis in the course of intrauterine fetal life may produce certain harmful effects on the developing brain of the fetus.


[The stereological study on normal and intrauterine growth retardation (IUGR) placentas]. The stereological study was carried out in 8 normal and 22 IUGR placentas through Image Analysis System of Cambridge Instruments Quantimet 970 and 10 indices were analysed. The results showed that in the IUGR group the placental indices of total surface area of acroteric villus, total volume of intervillous space, volume density of fetal capillaries, total volume of fetal capillaries and surface area density of vasculo-syncytial membrane were significantly reduced than those in the normal group. The vasculo-syncytial membrane became significantly thick in the IUGR placental group. It was confirmed that a close relation exists between IUGR and placental quantitative changes. The major pathological findings of the placenta were prematuration and hypoplasia.


[Clinical and pathological differential diagnosis of condyloma and pseudo-condyloma of vulva]. Three hundred and twenty-one biopsies from vulva, comprising 18 cases of condyloma acuminata (5.6%), 202 cases of pseudo-condyloma (62.9%), and 101 cases of non-specific vulvitis (31.5%), are reported. Clinically, most condylomata acuminata appeared as multiple soft papillary and verrucous lesions with local itchiness. The majority of patients with pseudo-condyloma were symptomless but with harbor roe-like warty papulae distributed symmetrically on both labia minora. Histologically, 18 cases of condyloma acuminata presented papillomatosis, parakeratosis, dyskeratosis, acanthosis, basal cell atypical hyperplasia and distinct koilocytosis. Pseudo-condylomata showed papillomatous growth but without atypical hyperplasia of basal cells and distinct koilocytosis.


[Effects of sex hormones on large bowel carcinogenesis induced by 1,2-dimethylhydrazine in Sprague-Dawley rats]. The effects of sex hormones on colorectal carcinogenesis induced by 1,2-dimethylhydrazine (DMH) were studied in Sprague-Dawley (SD) rats. The morbidity of large bowel carcinomas in group castration + estrogen (E2) + DMH (88%) was significantly higher than that in group male + DMH (56%), group castration+DMH (21%) and group castration+androgen + DMH (57%) respectively (P < 0.05, P < 0.01, P < 0.05). Part of tumors from those groups treated with DMH were analysed in detecting the possible presence of estrogen receptors(ER). Data showed that 3 out of 8 tumors (38%) in group castration + estrogen + DMH, 2 out of 7 (29%) in group male + DMH, 1 from 4 (25%) in group castration + DMH and 2 out of 6 (33%) in group castration + androgen + DMH contained E2-HRP receptor. The results also show that sex hormones, especially estradiol may inhibit ConA-induced lymphocyte transformation; promote cholesterol and bile metabolism and excretion in the liver; enhance pH value in colorectum and stimulate colonic epithelial proliferation which may elucidate the effects of sex hormones on colorectal carcinogenesis induced by DMH.


[Lipid peroxides, antiperoxidase and the progressive course of experimental atherosclerosis]. Rabbits were fed on cholesterol-rich (CHO) diet first for 60 days, and serum cholesterol, serum lipid peroxides (LPO) and superoxide dismutase (SOD) activities were measured at various periods after the withdrawal of the CHO-rich diet. The correlation of the above figures and the initiation, progression and regression of atherosclerotic lesion were studied. Results showed that: (1) when rabbits were fed on a CHO-rich diet for 60 days, the serum CHO and LPO increased significantly. Anyhow, the SOD activity was only slightly elevated when tiny atherosclerotic lesions developed in the aortae. (2) right after withdrawal of the CHO diet, serum CHO decreased significantly, but atherosclerotic lesion became even more severe than before. (3) after withdrawal of the CHO diet for 120 days, serum CHO, LPO content and SOD activity all returned to normal range and the atheromatous lesion became less severe except calcification and fibrosis developed in certain areas.


[Changes in type II alveolar epithelial cells in experimental allergic alveolitis]. Changes of type II alveolar epithelial cells (type II cells) were studied in experimental allergic alveolitis by using enzyme histochemical, electron microscopic and morphometric techniques. The results showed that the number of type II cells increased obviously after exposed to thermoactinomyces vulgaris (TV). The peak was on the 15th day and then decreased gradually till to normal on the 180th day. Type II cells could be divided into two populations in TV-treated rats, namely, the large type II cells and the small type II cells. The former ones bore a diameter larger than the value of mean + 2.58s and mean indicated the mean value of type II cells diameters of the controls. The large type II cells are known to be the hypertrophic cells.


[Investigation on the incidence of high altitude polycythemia and its hemoglobin characteristics in a Tibetan population]. The incidences of high altitude polycythemia (HAPC) and its hemoglobin characteristics in natives and immigrants at 2 different altitudes in the Tibet Autonomous Region were investigated. The results were as follows: 1) The incidences of HAPC among residents in Lhasa (3650 meters above sea level) and in Naqu-Ando district (4500-4800 meters above sea-level) were 2.39% and 12.95%, respectively. The incidence of HAPC increased with increasing altitude. The incidences in immigrants and in men were higher than those in natives and in women. 2) The MetHb concentration in RBC from patients with HAPC was 10.14%, distinctly higher than that in healthy adults (6.35%) in the same area. This may be one of the causes leading to oxygen carrying malfunction in the RBCs of HAPC patients. 3) The Mossbauer spectra of RBCs from patients with HAPC had a third component "c" in addition to the two normal components, oxy-Hb "a" and deoxy-Hb "b." The third component "c" may be adenatured Hb in the RBCs of the patients. The question of whether it is related to oxygen carrying power requires further study.


[The effects of dopamine D-1 and D-2 receptor antagonists on 3,4-dihydroxyphenylacetic acid (DOPAC) in rat striatum with differential normal pulse voltammetry]. Dopamine metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) was detected by differential normal pulse voltammetry (DNPV) in chloral hydrate anesthetized rat striatum. The effects of some dopamine D-1 and D-2 antagonists on DOPAC concentration were observed in order to demonstrate the role of presynaptic D-1 and D-2 receptors in the feedback of dopamine synthesis and metabolism. The results showed that the mixed antagonists haloperidol (0.5 mg/kg s.c.) and chlorpromazine (5 mg/kg s.c.), the selective D-1 antagonist SCH23390 (1 mg/kg s.c.), and the selective D-2 antagonist sulpiride (5 mg/kg s.c.) all increased DOPAC concentration significantly. The percentages of increase were 140%, 102%, 26% and 40% in comparison with basal DOPAC concentration (14 +/- 2 mumol/L), respectively. These results indicate that not only presynaptic D-2 receptor regulates DA synthesis and metabolism, but that the presynaptic D-1 receptor also takes part in this feedback mechanism.


[Primary hypothyroidism with obstructive sleep apnea syndrome]. The relationship between severity of hypothyroidism and OSA syndrome before and after treatment was analyzed and discussed. All patients had typical clinical features of primary hypothyroidism and were diagnosed by thyroid hormonal investigations. They were treated with conventional desiccated thyroid. Polysomnography and blood oxygen saturation % (SaO2%) were performed during sleep before and after substitutive treatment. In this series of 21 patients taking respiratory functional tests, 11 were consistent with OSA (42.4%). The serum values of TT4, FT4I, TT3 and TSH after treatment were markedly different from the initial values in 9 cases who were followed up after treatment (P < 0.001, respectively). The respiratory parameters including maximum apnea time, mean apnea time, respiratory disturbances index and the lowest oxygen saturation % were also markedly different in patients before and after treatment (P < 0.001-0.02, respectively). The serum levels of TT4 and FT4I were significantly correlated with the values of maximum and mean apnea time and the highest oxygen saturation % after substitution (r = 0.64-0.98, P < 0.01-0.05, respectively). We conclude that OSA in hypothyroidism showed the most satisfactory response to non-surgical treatment, and OSA syndrome could be cured as soon as hypothyroidism was controlled by desiccated thyroid.


[Purification, characterization and reverse cholesterol transport of human serum apolipoprotein A-IV]. In order to study the function of apoA-IV, human serum apoA-IV was isolated and purified. LDS was isolated with NaBr density gradient ultracentrifugation and then incubated with intralipid according to the method of Weinberg and Scanu. Protein complex containing apoA-IV was obtained. The apo-IV was purified from protein complex by preparative SDS-PAGE, electroelution and removal of SDS. ApoA-IV thus prepared gave a single band on SDS-PAGE with a MW of 46 kD. Amino acid composition and isoelectric focusing pI (5.27 and 5.47) of apoA-IV were similar to those reported in the literature. The effects of apoA-IV and liposome (apoA-IV: DMPC) on cholesterol efflux from hum skin fibroblast were studied. There was no significant difference (P > 0.5) between apoA-IV and control but a very significant difference (P < 0.001) between liposome (apoA-IV:DMPC) and control, suggesting that liposomes (apoA-IV:DMPC) play a role in reverse cholesterol transport. The results may offer a new approach for the prevention and treatment of atherosclerotic coronary heart disease.


[Androgen receptors in muscles mediate the trophic effect of testosterone on axotomized neurons]. In order to further explore the hypothesis that muscles are the primary action site for testosterone, the present study examined the effect of androgen receptor blockade in effector muscles on the efficacy of testosterone in preventing neuronal cell loss following axotomy. Hydroxyflutamide (HF), an androgen antagonist that acts competitively at the hormone receptor site, was administered into muscles of the tongue when animals were treated with testosterone following unilateral transection of the hypoglossal and facial nerves. The ability of testosterone to prevent neuronal cell loss in the axotomized hypoglossal and facial motor nucleus was examined. The results demonstrated that hydroxyflutamide completely negates the survival-promoting effect of testosterone on axotomized motoneurons.


[Distribution of type IV collagen, laminin and fibronectin during naris formation in the chick embryo]. The presence and distribution of extracellular matrix (ECM) macromolecules were analyzed in the facial primordia of chick embryos from stages (22-28) of development corresponding to naris formation. Frozen sections through the nasal placode, medial nasal processes and lateral nasal processes were prepared for indirect immunofluorescence employing a biotin-avidin DCS system using monoclonal antibodies against type IV collagen, laminin and fibronectin. The results demonstrated that ECM played a very important role in regulating migration, adhesion and anchorage of cells during morphogenesis. Strong fluorescence of basal lamina was present in the areas of rapid ingrowth of the nasal placode. It was suggested that ECM has very important guiding and regulating role during naris formation in chick embryo.


[Effects of EGF, anti-EGF and anti-EGFR antibodies on the growth of human cancer cells]. In this study, 3 human pancreatic adenocarcinoma cell lines (PC-1, PC-2 and PC-3) and 3 other human cancer cell lines (adenocarcinoma of lung, LETP; gastric carcinoma, SGL7901; and breast carcinoma, BCP37) were investigated by adding EGF, anti-EGF antiserum and anti-EGFR monoclonal antibody into culture medium. EGF was found to exert a mild stimulating effect on the growth of PC-1 and LETP cells, but had no effect on the other 4 cell lines. Anti-EGF and anti-EGFR antibodies inhibited the proliferation of PC-1, LETP and SGL7901 cells. No significant effect on the other 3 cell lines was seen. By using the Northern blot technique, expression of EGFR mRNA was identified in all 6 cell lines. There were 3 bands (10.5 kb, 5.8 kb and 2.8 kb) of EGFR mRNA in all cell lines except for LETP, in which the 10.5 kb band was absent. The results indicate that the effect of EGF on the growth of cancer cells is very complicated and may involve an unknown regulatory mechanism of cancer cell growth. EGF may exert stimulating or inhibiting effects on cancer cell proliferation, or it may have no effect at all, even though the EGFR gene was expressed in these cell lines.


[Analysis of natural history and long-term follow-up of 59 patients with obliterative pulmonary hypertension (OPH)]. The diagnosis of OPH was made by clinical and hemodynamic data in 59 patients, including 30 cases of unexplained pulmonary hypertension, 26 cases of pulmonary embolism and 3 others. The survival curve showed that the two-year survival rate of the patients was 76.72%, and the five-year rate was 63.37%. The single correlation factors influencing prognosis were the interval from onset of symptoms to diagnosis (course), cardiac function, PaO2, SaO2, cause of disease, pulmonary artery pressure and right atrial pressure. The outcome of long-term treatment (mean 4.2 years) in 15 patients with OPH was as follows: remarkable improvement in 8 cases, amelioration in 3, no change in 2 and deterioration in 2. The total effective rate was 73.33%. The importance of anticoagulant therapy and the use of nifedipine are discussed.


[Effects of dehydroepiandrosterone sulfate on the uterus and estrogen receptors in rats]. Dehydroepiandrosterone sulfate (DHA-S) at dosages of 50, 100 and 200 mg/kg s.c. for 3 days was shown to have obvious promotive effects on the growth of the uterus in immature rats. However, DHA-S given to ovariectomized rats at 40 and 100 mg/kg for 5 days was found to have no effect on the uterus. Incubation of granulosa cells for 3 h with DHA-S caused a significant increase in estradiol secretion. Even though DHA-S was found to increase uterine estrogen receptor concentration, this compound was shown to have no affinity for the estrogen receptor. It appears that the effects of DHA-S resulted mainly from the activity of its metabolite--estrogen.


[Study on L-form induction of C. albicans]. After culturing C. albicans with ketoconazole on high osmosing medium containing thioglycollate, pig serum and sucrose, a cell wall-deficient strain of C. albicans was obtained. We compared its physiologic and biochemical characteristics with those of the parent strain. This cell wall-deficient strain not only changed its morphology but also the chemical components of the cell wall. The cell wall-deficient strain lacked 4 characteristic protein bands and had cell wall mannan decreased to 44% of that of the parent strain. Animal models revealed differences between the toxicities of the two strains: the mean survival time of mice inoculated intravenously with 10(6) deficient strain cells was 23.5 days (70% of the mice died within 30 days), but that of mice injected with 10(6) parent strain cells was 15.8 days (100% of the mice died in 30 days).


[Grading of malignancy of thymomas by flow cytometry]. The nuclear DNA contents of paraffin embedded archival tissues from 33 cases of thymoma and carcinoma of the thymus were studied by flow cytometry (FCM). Twelve cases were ruled out because of coefficients of variance (CV) > 10.0, leaving 21 cases for analysis. Included were 9 cases of non-invasive thymomas and 12 cases of invasive thymomas and thymic carcinomas. Among non-invasive thymomas, the DNA content was diploid in 4 cases and aneuploid in 5 cases. The DNA content was aneuploid in all 12 cases of invasive thymomas and thymic carcinomas. Proliferative activity, determined by measuring S phase percentage and proliferative index, was significantly increased in invasive thymomas and thymic carcinomas as compared with the non-invasive ones. The average S phase percentage for invasive thymomas and thymic carcinomas was 29.17 +/- 6.91, and for non-invasive thymomas it was 15.86 +/- 3.17 (P < 0.001). The average proliferative indices in the invasive and non-invasive groups were 47.76 +/- 5.03 and 32.58 +/- 6.58, respectively (P < 0.001).


[Observation of the effect of PSTF oral liquor on the positive tuberculin test reaction]. The transfer factor is a medicine used for immunotherapy. It is mainly administered by injection. In this paper, the effect of PSTF oral liquor on immunity has been examined. Persons who have negative TB reaction were selected as the test sample. PSTF was taken orally for 4 weeks. Then the tuberculin test was done again. The test results indicated that in the test group the number of people who had a positive reaction and the size of the scleroma were obviously higher than those in the control group (P < 0.01, P < 0.001). In the test group with or without BCG vaccine scar, the ratio of positive reaction was higher than that in the control group (P < 0.05, P < 0.1). Within the test group the ratio of positive reaction in those with no scar did not differ from that in subjects without such scars (P > 0.05), so patients who have negative tuberculin reaction will have a higher ratio of positive reaction after taking PSTF orally for 4 weeks. This increased ratio can reach 53.33%. Thus, we conclude that the oral liquor is convenient and easily acceptable especially for the aged and children.


Genotoxic potential of tamoxifen and analogues in female Fischer F344/n rats, DBA/2 and C57BL/6 mice and in human MCL-5 cells. Chronic administration of tamoxifen to female rats causes hepatocellular carcinomas. We have investigated damage to liver DNA caused by the administration of tamoxifen to female Fischer F344/N rats or C57B1/6 or DBA/2 mice using 32P-postlabelling. Following the administration of tamoxifen for 7 days (45 mg/kg/day) and extraction of hepatic DNA, up to 7 radiolabelled adduct spots could be detected after PEI-cellulose chromatography of the 32P-labelled DNA digests. Tamoxifen caused a time-dependent increase in the level of adduct detected up to a value of at least 1 adduct/10(6) nucleotides after 7 days dosing. A dose response relationship was demonstrated over the range of 5-45 mg/kg/day (0.013-0.12 mmol/kg/day). On cessation of dosing there was a loss of adducts from the liver DNA. These adducts were not detected in DNA from vehicle-dosed controls or in DNA from kidney, lung, spleen, uterus or peripheral lymphocytes. Pyrrolidinotamoxifen caused a similar level of adduct formation as tamoxifen. In contrast, no significant adduct formation could be detected in liver DNA from rats given droloxifene or toremifene. Mice given tamoxifen (45 mg/kg/day for 4 days) showed levels of adducts in the liver which were 30-40% of those present in rats. Exposure of rat hepatocytes to tamoxifen in vitro, resulted in induction of unscheduled DNA synthesis, when preparations from rats which had been pretreated with tamoxifen in vivo were used. No such increase could be detected in hepatocytes from control rats, suggesting tamoxifen may induce enzymes responsible for its own activation. Tamoxifen induced a significant increase in micronucleus formation in a dose dependent manner in cultures of MCL-5 cells, a human cell line that expresses 5 different human cytochrome P450 isoenzymes, as well as epoxide hydrolase.


Effects of phenethyl isothiocyanate, a carcinogenesis inhibitor, on xenobiotic-metabolizing enzymes and nitrosamine metabolism in rats. Phenethyl isothiocyanate (PEITC), a constituent of cruciferous vegetables, has been shown to inhibit chemical carcinogenesis, possibly due to its ability to block the activation or to enhance the detoxification of chemical carcinogens. The present study was conducted to elucidate the biochemical mechanisms involved by characterizing the effects of PEITC on phase I and phase II xenobiotic-metabolizing enzymes. A single dose of PEITC to F344 rats (1 mmol/kg) decreased the liver N-nitrosodimethylamine demethylase (NDMAd) activity (mainly due to P450 2E1) by 80% at 2 h and the activity of NDMAd remained decreased by 40% at 48 h after treatment. The liver pentoxyresorufin O-dealkylase (PROD) activity and P450 2B1 protein level were elevated 10- and 7-fold at 24 h after treatment respectively. The liver microsomal ethoxyresorufin O-dealkylase (EROD) (mainly due to P450 1A) and erythromycin N-demethylase (mainly due to P450 3A) activities were decreased at 2-12 h after treatment and recovered afterwards. The lung microsomal PROD and EROD activities were not significantly affected; whereas, the nasal microsomal PROD and EROD activities were decreased by 40-50%. After a treatment with PEITC, the rates of oxidative metabolism of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) were decreased in liver microsomes by 40-60% at 2 h and recovered gradually; the rates in lung microsomes were markedly decreased by 60-70% at 2 h and remained at the decreased level at 24 h; and the rates in nasal mucosa microsomes were decreased gradually with the lowest activities observed at 18 h (50%) followed by a gradual recovery. Furthermore, the treatment with PEITC resulted in a maximal 5-fold increase of NAD(P)H:quinone oxidoreductase and 1.5-fold increase of glutathione S-transferase activities in the liver, but the activities of these two enzymes were not significantly affected in the lung and nasal mucosa. The sulfotransferase activity in the liver was decreased by 32-48% at 24-48 h after treatment; the nasal activity was increased by 1.8- to 2.5-fold, but the lung activity was not significantly changed. The hepatic UDP glucuronosyltransferase activity was slightly decreased at 2 h but slightly increased at 48 h after treatment, but no changes were observed for the lung and nasal activities. The study demonstrates that PEITC selectively affects xenobiotic-metabolizing enzymes in the liver, lung and nasal mucosa and it is especially effective in inhibiting the P450-dependent oxidation of NNK in the lung and of NDMA in the liver.


Chemical carcinogens and overnutrition in diet-related cancer. The intake of known dietary carcinogens was compiled and the cancer risk was estimated on the basis of carcinogenic potencies in animals as derived from the Carcinogenic Potency Database by Gold and co-workers. The total cancer risk was compared with the number of cancer cases attributed by epidemiologists to dietary factors (one-third of all cancer cases, i.e. approximately 80,000 per one million lives). Except for alcohol, the known dietary carcinogens could not account for more than a few hundred cancer cases. This was seen both with the DNA-reactive carcinogens (heterocyclic aromatic amines, polycyclic aromatic hydrocarbons, N-nitroso compounds, estragole, aflatoxin B1, ethyl carbamate, to name the most important factors) as well as with those carcinogens which have not been shown to react with DNA (e.g. caffeic acid and the carcinogenic metals arsenic and cadmium). Residues and contaminants turned out to be negligible. Among the various possibilities to explain the discrepancy we investigated the role of overnutrition. Dietary restriction in animals is well known for its strong reducing effect on spontaneous tumor formation. These data can be used to derive a carcinogenic potency for excess macronutrients: the tumor incidence seen with the restricted animals is taken as a control value and the increased tumor incidence in the animals fed ad libitum is attributed to the additional feed intake. For excess standard diet in rats, a carcinogenic potency TD50 of 16 g/kg/day was deduced from a recent study. Overnutrition in Switzerland, estimated to be 5.5 kcal/kg/day, was converted to excess food (1.9 g/kg/day) and the cancer incidence was calculated. The result, 60,000 cancer cases per one million lives, is provocatively close to the number of cases not explained by the known dietary chemical carcinogens. Mechanistic studies will be required to test our hypothesis and investigate the role of different types of macronutrients in overnutrition.


Cell kinetic analysis of the mucosal epithelium and assay of ornithine decarboxylase activity during the process of 1-hydroxyanthraquinone-induced large bowel carcinogenesis in rats. Cell kinetics and activity of ornithine decarboxylase (ODC) were studied during the process of 1-hydroxyanthraquinone (1-HA)-induced intestinal carcinogenesis in rats. Starting at 6 weeks of age, a total of 37 male ACI/N rats were divided into two groups and treated as follows: group I (18 rats) received diet containing 1% 1-HA for 12 months; group II (19 rats) was given the basal diet alone. Sub-groups of 5-7 rats were sequentially killed at 4, 8 and 12 months for evaluation of the length, cell numbers and 5-bromo-2'-deoxyuridine (BrDU) labeling indices of large bowel crypts together with ODC activity. All kinetic and ODC data indicated increased DNA synthesis and proliferation at all time points. Morphological observation of the intestines also revealed melanosis, crypt abscesses and erosion, becoming more pronounced with length of exposure to the anthraquinone. The data thus suggest that cell proliferation in the crypts of the cecum or colon is important for 1-HA-induced intestinal carcinogenesis.


N-hydroxy-MeIQx is the major microsomal oxidation product of the dietary carcinogen MeIQx with human liver. 2-Amino-3,8-dimethylimidazo[4,5-f]quinoxaline (MeIQx), one of the most abundant of the heterocyclic aromatic amines formed during the cooking of meat, is genotoxic and carcinogenic in rodents. MeIQx requires metabolic activation by P450 before it can exert these effects. Whilst there is indirect evidence that the mutagenic product is N-hydroxy-MeIQx (N-OHMeIQx), we have now identified this unequivocally following incubation of the amine with human hepatic microsomal fraction. A mixture of unlabelled MeIQx, [13C,15N2]MeIQx and [14C]MeIQx was used as substrate and the products analysed by HPLC-thermospray mass spectrometry. Characteristic doublet ions, 3 mass units apart, were found at m/z 214/217 ([M+H]+) from the parent compound, MeIQx and at 230/233 ([M+H]+) from N-OHMeIQx. The presence of a doublet ion at m/z 214/217 with the doublet at 230/233 [M+H+] provided additional evidence that this was N-OHMeIQx, as facile loss of 'O' is characteristic of N-hydroxylamines. Further evidence for the identity of the major metabolite, which accounted for approximately 90% of all microsomal metabolism, was obtained by comparing the mutagenicity of the HPLC eluate using Salmonella typhimurium YG1024, which is particularly sensitive to N-hydroxylamines, and TA98/1,8-DNP6 which is resistant to most N-hydroxylamines. Ninety-five per cent of direct-acting mutagenicity present in the reaction mixture was associated with a single peak, which co-eluted with N-OHMeIQx, as indicated by mass spectrometry. In the presence of a metabolic activation system, only one additional mutagenic peak, corresponding to unchanged MeIQx, could be detected. MeIQx (5 microM) was N-hydroxylated at a rate of 77 +/- 11 pmol/mg/min (mean +/- SEM, n = 4) by human liver microsomes. The specific inhibitor of human CYP1A2, furafylline (5 microM) inhibited the N-hydroxylation of MeIQx by > 90%. These data show that N-OHMeIQx is both the major oxidation product and the major genotoxic product of MeIQx generated by microsomal fractions of human liver and that the reaction is catalysed almost exclusively by CYP1A2.


Metabolism of 1- and 2-naphthylamine in isolated rat hepatocytes. The liver probably plays a major role in the metabolic activation of the bladder carcinogen 2-naphthylamine (2-NA) and in the inactivation of the non-carcinogenic isomer 1-naphthylamine (1-NA). However, metabolic profiles of these compounds (including primary metabolites and directly determined conjugates) in hepatocytes are not available. Therefore metabolism of 1- and 2-NA was compared in freshly isolated hepatocytes from 3-methylcholanthrene (MC)-treated and untreated rats. At 10 microM, 2-NA was found to be mainly N-acetylated (66% of total metabolites after 1 h incubation) and N-glucuronidated (19%). Minor pathways led to C-oxidation (7%) and N-oxidation (3%; 2% present as the N-glucuronide). In hepatocytes from MC-treated rats total metabolism was slightly affected (1.5-fold increase). However, C- and N-oxidation were markedly increased (63 and 18% respectively), while N-acetylation and N-glucuronidation were diminished (5 and 2% respectively). Similar experiments were carried out with 1-NA. Its N-glucuronide was the predominant metabolite (68%) followed by the N-acetylated compound (15%) while C-oxidation was low and N-oxidized metabolites could not be detected, even after induction. The results demonstrate that MC treatment markedly shifted 2-NA metabolism from N-acetylation and N-glucuronidation to N- and C-oxidation. In the case of 1-NA metabolism extensive N-glucuronidation together with the lack of N-oxidation may prevent carcinogenesis.


Oxidative DNA damage estimated by 8-hydroxydeoxyguanosine excretion in humans: influence of smoking, gender and body mass index. Oxidative DNA damage may be implicated in ageing, carcinogenesis and other degenerative diseases. Oxidative DNA damage can be assessed in humans in vivo from the urinary excretion of the DNA-repair product 8-hydroxydeoxyguanosine (8OHdG). We investigated factors influencing the excretion of 8OHdG in 24 h urine from 83 randomly selected healthy subjects (52 women) aged 40-64 years. For 2 weeks prior to urine collection the subjects kept a weighed diet record. 8OHdG was quantified by an automatic three-dimensional HPLC analysis with electrochemical detection. The 8OHdG excretion was 252 +/- 103 (mean +/- SD) pmol kg body weight/24 h with a range from 78 to 527. Multiple regression analysis identified three factors, smoking, body mass index (BMI) and gender, as significant predictors of the 8OHdG excretion. In 30 smokers the 8OHdG excretion was 320 +/- 99 pmol/kg/24 h opposed to 213 +/- 84 pmol/kg/24 h in 53 non-smokers. According to multiple regression analysis smokers excreted 50% (31-69%; 95% confidence interval) more 8OHdG than non-smokers. In 52 women the 8OHdG excretion was 240 +/- 106 pmol/kg/24 h opposed to 271 +/- 96 pmol/kg/24 h in 31 men. According to the multiple regression analysis men excreted 29% (10-48%) more 8OHdG than women. According to multiple regression analysis the 8OHdG excretion decreased with 4% (2-6%) per increment in BMI measured in kg/m2. The dietary distribution of energy demonstrated no important predictive value with respect to 8OHdG excretion. The intake of the antioxidant vitamins C and E and of vitamin A equivalents, including beta-carotene, was not associated with 8OHdG excretion. The results suggest that smoking increases oxidative DNA damage by approximately 50%. This effect implies potential serious health effects adding to the other well-known health hazards of smoking. The higher 8OHdG excretion in men and lean subjects may be related to a higher rate of metabolism with increased availability of reactive oxygen species. The apparent 7-fold individual variation in oxidative DNA damage carries implications regarding the rate of ageing and the risk of cancer and other degenerative diseases. The excretion of 8OHdG into urine offers a valuable tool for testing such hypotheses in humans.


Modulation of glutathione S-transferases and glutathione peroxidase by the anticarcinogen butylated hydroxyanisole in murine extrahepatic organs. Induction of glutathione S-transferases (GST) by the anticarcinogen butylated hydroxyanisole (BHA) has been examined in lung, kidney and small intestine of male and female BALB/c mice. BHA produced maximal induction of GST in the gut and although it increased GST levels in the kidney, it had little effect on pulmonary GST. Dietary BHA induced Alpha (Ya and Yk), Mu (Yb) and Pi (Yf) class GST subunits at least 10-fold in the small intestine but, by contrast, selenium-dependent glutathione peroxidase activity was reduced by approximately 4-fold in this organ following BHA treatment. In the kidney, all of the GST subunits, apart from Yk in males, showed modest levels of induction by BHA. However, a pronounced sex difference in the expression of renal alpha class subunits in both control and BHA-treated mice was observed, with female mice expressing approximately 4-fold greater levels of Ya and Yk than male mice. All renal GST were localized primarily in the proximal tubules. Dietary BHA was found to have the least inductive effect in the lung, where the GST were localized solely in the bronchi. The pulmonary Mu class GST subunits were induced approximately 2-fold by BHA; the expression of other GST was marginally increased by this inducer. Alpha class GST was also subject to sexual differentiation in the lung with female mice possessing higher levels of Yc and Yk than males. The Ya-type subunit was not detected in the lung nor was it induced by BHA.


Binding of the food mutagen PhIP in pigmented tissues of mice. The distribution of the 14C-labelled food mutagen 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in the tissues of C57B1/6 and NMRI mice was studied. The results showed a high and selective binding of radioactivity in the pigment epithelium of the eye and in the fur following a single dose (0.3-4 mg/kg) of [14C]PhIP in the pigmented C57B1/6 mice whereas no such localization of radioactivity was present in the albino NMRI mice. A low but selective covalent binding of radioactivity was observed in the liver, inner cortex of the kidney and in the tracheal mucosa of [14C]PhIP-injected mice. PhIP was firmly bound to synthetic melanin pigment in vitro; only 3% was released by extraction with a phosphate buffer (pH 7) whereas 72% was released by extraction by methanol:conc. NH3 (15:1). Three hours to 7 days following a single injection of [14C]PhIP in C57B1/6 mice the radioactivity in the eye was 3- to 6-fold higher than that in the liver or kidney. Almost 60% of the radioactive material present in the pigmented epithelium of the eye 3 and 24 h following injection could be extracted by basic methanol and identified as unchanged PhIP. The residual radioactivity in the pigmented epithelium of the eyes may represent a covalent binding of [14C]PhIP metabolites to cellular constituents or to a basic methanol-resistant binding of [14C]PhIP to melanin. The results indicate that pigmented tissues may be potential target tissues for the toxic effects of PhIP and suggest that the use of hair for biological monitoring of PhIP should be examined.


Comparative tumorigenicity of nitrochrysene isomers in newborn mice. 6-Nitrochrysene (6-NC) is a potent lung and liver carcinogen in the newborn mouse assay. In this report, we extended our studies of the structure--tumorigenicity relationships of the mononitrochrysene isomers. We synthesized 1-NC, 2-NC and 3-NC by oxidation of the corresponding aminochrysenes with mCPBA; efforts to synthesize 4-NC and 5-NC from 4- and 5-aminochrysene were not successful. The tumorigenic activities of 1-NC, 2-NC, 3-NC and 6-NC were compared. Groups of mice were treated with the appropriate compounds in dimethylsulfoxide (DMSO) by i.p. injection on the 1st, 8th and 15th day of life. At a total dose of 100 nmol/mouse, 6-NC induced significant incidences and multiplicities of lung tumors in mice in both sexes; only males were susceptible to liver tumor induction. At 100 nmol/mouse, induction of lung and liver tumors by 1-NC, 2-NC and 3-NC was not significantly different from that observed in mice treated with DMSO. The results indicate that nitro substitution at the 6-position of chrysene is critical for strong tumorigenicity in the newborn mouse assay.


Influence of fruit and vegetable juices on the endogenous formation of N-nitrosoproline and N-nitrosothiazolidine-4-carboxylic acid in humans on controlled diets. Water, green pepper, pineapple, tomato, strawberry, carrot, and celery juices were made 46 mg/100 ml in ascorbic acid by the addition of distilled water or ascorbate. The ability of each juice to inhibit endogenous formation of N-nitrosoamino acids (NAA) in humans was determined in controlled experiments. Sixteen men consumed a standard diet low in nitrate and ascorbic acid for 18 consecutive days. Nitrate (5.24 mmol) and L-proline (4.35 mmol) were given orally on days 3, 5, 7, 9, 11, 13, 15, 17 and 18. On days 3 and 18, L-proline was immediately followed by 100 ml distilled water (positive control). On days 5, 7, 9, 11, 13, 15 and 17, L-proline was immediately followed by 100 ml juice or 46 mg ascorbate in 100 ml distilled water (treatment). Only diet was given in between dosing days to ensure baseline levels of NAA excretion. Urine was collected for 24 h following treatments and analyzed for NAA. Green pepper, pineapple, tomato, strawberry and carrot treatments significantly inhibited N-nitrosoproline (NPRO) formation relative to the positive control. Also, green pepper, pineapple and tomato juices significantly inhibited NPRO formation relative to ascorbic acid alone. Green pepper significantly inhibited N-nitrosothiazolidine-carboxylic acid formation relative to ascorbic acid alone. These data demonstrate that green pepper, tomato, pineapple, strawberry and carrot juice have greater ability to inhibit endogenous nitrosation than would be expected based solely on their ascorbate content.


Absence of ras oncogene activation in rat urinary bladder carcinomas induced by N-methyl-N-nitrosourea or N-butyl-N-(4-hydroxybutyl)nitrosamine. Previously, we demonstrated point mutations of the H-ras gene in N-[4-(5-nitro-2-furyl)-2-thiazolyl]formamide (FANFT)-induced rat urinary bladder carcinomas. In this study, ras oncogene activation was examined in urinary bladder carcinomas induced by N-(4-hydroxybutyl)nitrosamine (BBN) or N-methyl-N-nitrosourea (MNU) administration followed by uracil treatment. In the first experiment, MNU (20 mg/kg body wt) was i.p. injected into 11 male F344 rats twice a week for 4 weeks, followed by feeding 3% uracil for 20 weeks (MNU/uracil group). Ten rats were given only 3% uracil without MNU pretreatment. In the second experiment, 20 male F344 rats were given 0.05% BBN in the drinking water for 4 weeks, then fed 3% uracil for 20 weeks (BBN/uracil group). Another 20 rats were fed 3% uracil without the BBN pretreatment. Transitional cell carcinomas were induced in the urinary bladder of all rats in the MNU/uracil and BBN/uracil groups. Papillomas and hyperplasias were present in the rats given uracil without prior BBN or MNU. DNA and protein were extracted from the tumors (MNU/uracil or BBN/uracil groups) or from the scraped bladder epithelium (uracil alone groups). Sequences around codons 12, 13 and 61 of H-, K- and N-ras genes were examined by direct sequencing after polymerase chain reaction, and p21 was examined by Western blotting. No mutation was found within the examined sequences and p21 showed no changes in mobility. There was no difference in the level of p21 expression between rats treated with MNU/uracil or BBN/uracil compared to corresponding uracil alone groups. These results indicate that the ras oncogene was not activated in urinary bladder carcinomas induced by BBN or MNU in combination with uracil treatment, in contrast to previous findings with FANFT.


Inhibition of DNA synthesis by phenobarbital in primary cultures of hepatocytes from normal rat liver and from hepatic nodules. One of the many hypotheses put forward to explain the mechanism by which phenobarbital (PB) promotes hepatocarcinogenesis is by differential mitoinhibition of surrounding hepatocytes while allowing the initiated hepatocytes to respond to growth stimuli and form foci and nodules. Given the similarity in structures between PB and orotic acid (OA), another rat liver tumor promoter, the present investigation was designed to determine (i) whether PB, like OA, exerts its mitoinhibitory effect at a site beyond the growth factor receptor and receptor mediated early events; and (ii) whether PB exerts a differential mitoinhibitory effect by selectively inhibiting the non-initiated hepatocytes but not the initiated hepatocytes in vitro. Our studies demonstrate that, like OA, PB also inhibits DNA synthesis in hepatocytes from normal rat liver in a dose dependent manner with 80-90% at a dose of 6 mM. One target site may lie beyond the growth factor receptor mediated early events because PB inhibited DNA synthesis in hepatocytes primed with the growth factor 24 h earlier. Interestingly, PB inhibited DNA synthesis not only in hepatocytes from non-nodular surrounding liver but also in hepatocytes from persistent hepatic nodules initiated with 1,2-dimethylhydrazine and promoted with OA. Therefore, our results suggest that although PB is a mitoinhibitor of DNA synthesis in hepatocytes, it does not appear to create as strong a differential mitoinhibition between non-nodular surrounding and initiated hepatocytes as is evident in the resistant hepatocyte and OA models. These results raise the question whether differential mitoinhibition is the major contributing factor in the PB mediated rat liver tumor promotion.


The insecticide endosulfan and its two stereoisomers promote the growth of altered hepatic foci in rats. The non-genotoxic, chlorinated cyclodiene insecticide endosulfan was studied for its ability to act as a tumour promoter in a two-stage, altered hepatic foci bioassay in male Sprague-Dawley rats. Two stereoisomers of endosulfan, alpha-endosulfan (ENDO alpha) and beta-endosulfan (ENDO beta) were used, as well as a commercially-occurring mixture of the alpha- and beta-isomers (ENDO alpha beta). The animals were initiated by intraperitoneal injection of nitrosodiethylamine 24 h after a two-thirds-partial hepatectomy. Five weeks later the animals were transferred to diets containing 30, 100 and 300 p.p.m. of either ENDO alpha beta, ENDO alpha or ENDO beta. The study was terminated 25 weeks after initiation and the development of foci of gamma-glutamyltranspeptidase-positive hepatocytes was evaluated by stereological methods. The results show that endosulfan and its two stereoisomers promote the development of altered hepatic foci, suggesting that endosulfan is a tumour-promoting agent acting by clonal expansion of initiated cells.


A possible explanation for the differential cancer incidence in the intestine, based on distribution of the cytotoxic effects of carcinogens in the murine large bowel. The ability of four mutagenic/carcinogenic chemicals administered as single doses to induce a programmed form of cell death (apoptosis) in the BDF1 mouse large bowel was studied and compared with a previous study on the small intestine using the same mice. The number of apoptotic cells was counted following treatment with the direct-acting agents N-nitroso-N-methylurea (NMU) and N-nitroso-N-ethylurea (NEU) and two agents which require metabolic activation 1,2-dimethylhydrazine (DMH) and N-nitrosodimethylamine (NDMA). DMH (80 mg/kg) was the most effective at inducing acute cell death and this was closely followed by NMU (200 mg/kg). The least effective agent in the large bowel was NDMA. The peak yield of apoptosis occurred between 4 h (NEU) and 8 h (DMH) after treatment. An analysis of the changing shapes of the frequency plots of apoptosis at each cell position in the crypt at various times after exposure permits an estimate to be made of the position in the crypt of the primary target cells for the cytotoxic action at time t = 0. For the agents studied, this is in the range of the 5th to the 10th position from the base of the crypt. This distribution for the target cells for apoptotic cell death is not coincident with that for the presumptive stem cells, which is at cell position 1 or 2. Comparisons with results previously obtained in the small intestine (ileum) of the same mice show that the relative cytotoxic effectiveness of the four agents differs. Furthermore, the position of the target cells is at about the 4th position from the bottom of the crypt in the ileum, and here the distribution is coincident with that presumed for the stem cells. Our interpretation of the data is that damaged cells in the stem cell region of the small bowel are removed by the activation of a cell suicide programme, which effectively removes potentially harmful genetic alterations. In contrast, in the large bowel, cell death is not initiated particularly strongly in the stem cell region but tends to occur higher in the crypt. The absence of this selective deletion process may result in the perpetuation of deleterious mutations in the colonic stem cell population and this may explain in part, the higher incidence of cancers observed in the large bowel.


Duodenal reflux through the pylorus induces gastric adenocarcinoma in the rat. We investigated whether duodenal reflux through the pylorus is involved in the development of gastric cancer. Male Wistar rats weighing 230-250 g were subjected to three types of operative procedures: (i) allowing reflux through the pylorus; (ii) allowing reflux through a gastrojejunal stoma; and (iii) gastrotomy. No carcinogens were given, and the animals were killed 50 weeks after surgery. No cancers were detected in any of the 18 animals with gastrotomy. In contrast, seven (41%) of 17 animals with reflux through the pylorus and four (31%) of 13 animals with reflux through the stoma had adenocarcinoma. Differences in the incidence between both reflux groups and the gastrotomy group were significant (P < 0.01 and P < 0.05 respectively). All of the adenocarcinomas developed in the pyloric mucosa near the pylorus in the animals with reflux through the pylorus, and in the oxyntic mucosa near the stoma in those with reflux through the stoma. Adenocarcinomas appeared as a polyploid mass with or without slight central erosion. Most of the adenocarcinomas were of the well-differentiated tubular type, and the others were of the mucinous type. No differences in either the histologic type or depth of invasion of the adenocarcinoma were recognized between the two duodenogastric reflux groups. Precancerous or paracancerous lesions, such as adenoma, adenocystic proliferation, and stomal pseudopyloric metaplasia, were more frequently found in the same region as the adenocarcinomas. These findings suggest that duodenogastric reflux in the rat has potent carcinogenic activities not only in the oxyntic mucosa through the stoma, but also in the pyloric mucosa through the pylorus.


Substances in human urine that strongly inhibit bacterial mutagenicity of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) and related heterocyclic amines. Extracts of human urine were shown to contain substances that strongly inhibited the liver S9-mediated mutagenicity of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP) in Salmonella typhimurium TA98 strain in a liquid incubation assay. The inhibitory effect was unrelated to cytotoxicity and was similar with urine extracts from smokers and non-smokers. Under similar assay conditions, the mutagenicity of the related amino-imidazoazaarenes, 2-amino-3-methyl-imidazo[4,5-f]quinoline, 2-amino-3,8-dimethylimidazo[4,5-f]-quinoline and 2-amino-3,8-dimethylimidazo[4,5-f]quinoxaline was also found to be strongly inhibited by urine extracts. Decreased or enhanced mutagenicity was seen with 2-acetyl-aminofluorene and 2-aminoanthracene depending on the type of assay, and the time of incubation in liquid medium. A weak inhibition of the mutagenicity of 2-nitrofluorene, a direct-acting mutagen, was observed only after a short incubation time. Mutagenicity of 4-nitroquinoline N-oxide was not altered by the presence of urine extracts at concentrations shown to be inhibitory for the mutagenicity of heterocyclic aromatic amines. Our data suggest that the inhibitory substances in urine act through their capacity to non covalently bind the parent heterocyclic and aromatic amines, thus affecting their availability in aqueous medium for diffusion into liver microsomes where metabolic activation takes place.


Sequential changes in glycogen content, expression of glucose transporters and enzymic patterns during development of clear/acidophilic cell tumors in rat kidney. Renal clear cell tubules and clear/acidophilic cell tumors were induced in male Sprague-Dawley rats by 7 weeks oral administration (stop model) of N-nitrosomorpholine (NNM) at a concentration of 12 mg/100 ml in the drinking water. Twelve, 23 and 34 weeks after withdrawal of NNM serial cryostat sections of the kidneys were histochemically analyzed for the following parameters: glucose transporter proteins (GLUT1, GLUT2), glycogen content and the activities of glycogen synthase (SYN), glycogen phosphorylase (PHO), glucose-6-phosphatase (G6Pase), glucose-6-phosphate dehydrogenase (G6PDH), hexokinase (HK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), pyruvate kinase (PK), succinate dehydrogenase (SDH), malate dehydrogenase (MDH), alkaline phosphatase (ALP), acid phosphatase (ACP) and gamma-glutamyltransferase (GGT). Clear cell (glycogenotic) tubules first appeared at 23 weeks, and clear/acidophilic cell tumors at 34 weeks after withdrawal of the carcinogen. G6Pase, ALP, GGT and GLUT2 were absent in clear cell tubules, clear/acidophilic cell tubules, and clear/acidophilic cell tumors indicating a sequential origin of all these types of lesions from the collecting duct system, in line with previous morphological findings. In comparison to the collecting duct epithelium, glycogenotic tubules demonstrated an increased activity of PHO and reduced activities of glycolytic and mitochondrial enzymes, which were accompanied by a strongly reduced expression of GLUT1. Moderately increased activities of glycolytic and mitochondrial enzymes were observed in the clear cells of clear/acidophilic cell tubules and tumors compared with those in glycogenotic tubules. They had slightly increased activities of the glycolytic enzymes GAPDH and PK compared with normal collecting duct epithelium, while most of them were nearly lacking in GLUT1. Our findings suggest that glycogen storage is not due to an increased uptake of glucose from the blood, but results from a disturbance in intracellular flux of metabolites. The development of clear cell tubules from the normal collecting duct epithelium is accompanied by a markedly decreased expression of GLUT1 along with a reduction in glycolytic and mitochondrial enzymes. This reduction of enzyme activities is replaced by an increase in enzyme activities in clear/acidophilic cell tumors indicating a fundamental shift in carbohydrate metabolism during progression from preneoplastic to neoplastic lesions.


Analysis of the binding sites of chromium to DNA and protein in vitro and in intact cells. Previous studies have examined Cr(III), or CrO4 reduced to Cr(III), binding in vitro to DNA. However, there have been few studies examining chromate binding to DNA in intact cells. Treatment of intact cells with chromate (Na2(51)CrO4) resulted in chromium (Cr) binding to DNA. The binding of Cr to DNA was much more stable when more residual peptide/amino acids were associated with DNA. A substantial portion of the Cr bound to DNA was released by treatment with EDTA, suggesting that trivalent Cr was the major oxidation state of Cr bound to DNA. Cr(III) stimulated the formation of amino acid-DNA and protein-DNA complexes in vitro. Tyrosine and cysteine exhibited the highest activity in being complexed to DNA by Cr(III) in vitro, while histidine, methionine and threonine also exhibited more activity than any other amino acid. Similar results were found in intact cells. The activity of proteins complexed to DNA by trivalent Cr depended upon the content of these reactive amino acids. Thus, bovine serum albumin was more active than actin, which in turn was more active than histones. These and other studies presented suggested that Cr(III) was involved directly in the formation of DNA-protein complexes in intact cells, unlike other metals such as Ni(II), which are thought to form DNA-protein cross-links catalytically and not participate directly in the complex. The majority of trivalent Cr associated with DNA was bound to the phosphate backbone without exhibiting any base specificity. Collectively, these results indicate that trivalent Cr creates DNA protein crosslinks by binding with reactive amino acids (i.e. cysteine, tyrosine or histidine) and linking these to the phosphate backbone of DNA.


DNA polymerase action on benzo[a]pyrene-DNA adducts. A 16mer oligonucleotide containing a single guanine residue at nucleotide 13 from the 3' end was treated with the (+)-enantiomer of the 7,8-dihydrodiol 9,10-epoxide of benzo[a]pyrene (B[a]P). Oligonucleotides containing either an adduct in which the epoxide ring was opened trans or cis by the amino group of the guanine residue were separated by chromatography and identified by 32P postlabeling and circular dichroism spectroscopy. In the presence of nucleotide triphosphates and DNA polymerase (either Sequenase, version 2.0 or human polymerase alpha), it was found that the B[a]P adducts inhibited extension of an 11mer primer opposite the nucleotide 3' to the adduct in the template. Under various conditions, this inhibition was greater for the cis adduct than for the trans adduct. After a 10 min incubation with Sequenase, primer extension was reduced to approximately 20% of that seen with unmodified oligonucleotide by the trans adduct and was almost completely inhibited by the cis adduct. When a 12mer primer was used to examine nucleotide incorporation directly across from the guanine or adducted guanine residues, it was clear that deoxycytidylic acid was preferentially incorporated in all cases but that the incorporation was severely inhibited by both the cis and trans adducts. These findings suggest that a cis adduct is a more effective block to replication than a trans adduct, and that these adducts may not be very efficient mutagenic lesions.


Entrapment by magnetic microcapsules of the protein pyrolysates IQ, PhIP and Glu-P-1, and alteration of IQ metabolite exposure within the rat gastrointestinal tract by risk-modulating components of the human diet. The entrapment of heterocyclic aromatic amine gastrointestinal (GI) carcinogens (HAAs), by retrievable semipermeable magnetic polyethylenimine (PEI) microcapsules was investigated in vitro and in vivo as an approach for human biomonitoring. Previous studies showed that PEI microcapsules successfully entrapped benzo[a]pyrene (B[]P) and its metabolites in the GI tract of rodents. In this study, we have shown that 14C-labelled 2-amino-3-methylimidazo[4,5f]quinoline (IQ), 2-amino-1-methylphenylimidazo[4,5-b]pyridine (PhIP) and 2-amino-6-methyldipyrido[1,2-a:3'2'-d]imidazole (Glu-P-1) are adsorbed to PEI microcapsules in vitro and can be desorbed by treatment with ammoniac methanol. Binding of HAAs to PEI microcapsules containing copper phthalocyanine (TCPTS), a moiety which reversibly binds chemicals with aromatic planar structures, was 2- to 4-fold higher than with unmodified PEI microcapsules. PEI microcapsules also acted as a nucleophile and trapped the proximate carcinogenic metabolite of IQ, 2-hydroxy-amino-3-methyl-imidazo[4,5f]quinoline (N-hydroxy-IQ). The entrapment of 14C-labelled IQ and PhIP by microcapsules was investigated in vivo in male F344 rats fed a conventional chow diet or a human diet with varying amounts of fat and beef intake typically consumed in the UK. Animals were adapted to human diets which were either high (H) or low (L) in fat (F), beef protein (B) and dietary fibre non-starch polysaccharide (NSP). Microcapsule entrapment of IQ and metabolites was 0.5-2.0% of the dose and 4-fold higher in rats consuming a HF/HB/LNSP than those consuming a LF/LB/HNSP diet, these being respectively putatative high- and low-risk-associated diets. In the HF/HB/LNSP diet group, a higher amount of IQ metabolites were detected in the microcapsules; a lower proportion of covalently bound metabolites could be removed by acid hydrolysis. Urinary excretion was 2-fold greater and analysis of the urinary metabolites showed there to be lower sulfotransferase activity than in the LF/LB/HNSP group. The amount of 14C-labelled PhIP entrapped by PEI microcapsules was 1.5% of the dose in rodents fed a LF/HB/LNSP human diet and binding was 7-fold higher than in rodents fed a semi-purified diet. These results demonstrate that microcapsules can entrap IQ and PhIP and their metabolites within the GI tract of rodents. The amounts entrapped by microcapsules in the rodent model suggests that this approach may be feasible for human biomonitoring of HAAs and for non-invasively studying dietary modulations of carcinogen exposure within a potential HAA target organ at high risk from as-yet unidentified causes.


Characterization of thymidine adducts formed by acrolein and 2-bromoacrolein. Thymidine was permitted to react with the known mutagens acrolein and 2-bromoacrolein under physiological conditions. The products of these reactions were separated by HPLC and characterized by UV, FAB/MS, electrospray MS, 1H NMR and chemical transformation. The reaction with acrolein gave one major product, N3-(3''-oxopropyl)thymidine, which is unstable in aqueous solution and was reduced with sodium borohydride to the corresponding alcohol. Reaction with 2-bromoacrolein yielded the unstable intermediate, N3-(2''-bromo-3''-oxopropyl)thymidine, and two stable products, the diastereomers of N3-(2''-hydroxy-3''-oxopropyl)thymidine, which are slowly transformed to N3-(2''-oxo-3''-hydroxypropyl)thymidine. Reactions with both mutagens proceed most rapidly at pH 9.2, less rapidly at pH 7.4, and no products are found at pH 4.2. Stable adducts found in the reaction of 2-bromoacrolein were also identified in reactions with single-strand oligodeoxynucleotides using a sensitive, selected ion monitoring GC/MS procedure.


Analysis of phospholipid metabolism in murine keratinocytes transformed by the v-ras oncogene: relationship of phosphatidylinositol turnover and cytokine stimulation to the transformed phenotype. Introduction of a v-rasHa oncogene into cultured mouse keratinocytes by transduction with a defective retrovirus is sufficient to transform keratinocytes to the benign phenotype. Transduced keratinocytes overexpress TGF alpha and hyperproliferate in culture medium with 0.05 mM Ca2+. Whereas normal keratinocytes respond to elevated medium Ca2+ by cessation of proliferation and induction of terminal differentiation, v-rasHa keratinocytes are not induced to differentiate by Ca2+. We now demonstrate that v-rasHa keratinocytes have elevated basal levels of phosphatidylinositol, inositol phosphates and diacylglycerols in 0.05 mM Ca2+ medium. Basal turnover of phosphatidylcholine is not altered by the rasHa oncogene. The generation of inositol phosphates is even further stimulated in v-rasHa cells by an increase in extracellular Ca2+ or by exposure to aluminum fluoride. Thus, the v-rasHa gene product does not stimulate the inositol phospholipid pathway maximally and additional phosphatidylinositol is available for turnover in response to inducers of phospholipase C activity. TGF alpha and medium conditioned by v-rasHa keratinocytes, both of which stimulate proliferation of normal cells in 0.05 mM Ca2+, transiently increased phosphatidylinositol turnover in normal keratinocytes but did not inhibit Ca(2+)-induced terminal differentiation. In contrast, sustained elevation in basal phosphatidylinositol metabolism was produced by aluminum fluoride. Combined exposure to aluminum fluoride and exogenous TGF alpha caused hyperproliferation, resistance to Ca(2+)-induced differentiation and morphological changes identical to those of v-rasHa keratinocytes. These results provide a link between the biological consequences of v-rasHa gene expression and biochemical changes which are known to alter the keratinocyte phenotype.


Changes in adult metabolism of aflatoxin B1 in rats neonatally exposed to diethylstilbestrol. Alterations in alpha-class glutathione S-transferases. Neonatal exposure of rats to xenobiotics has been shown to produce long-term alterations in hepatic enzyme activities and in levels of DNA adducts following carcinogen exposure. We exposed newborn male rats to diethylstilbestrol (DES), pregnenolone-16 alpha-carbonitrile, 7,12-dimethylbenz[a]anthracene or phenobarbital on days 1, 3 and 5 of age. At five months of age, males were injected with 1 mg/kg of [3H]aflatoxin B1 (AFB1), killed after 2 h and examined for AF-DNA adduction in the liver. Males neonatally exposed to DES showed a 35% decrease in DNA adduction levels. Analysis of the adducted DNA bases failed to show any changes in relative proportions of individual adducts in the DES samples compared to controls. Hepatic glutathione concentrations were unchanged. However, Western blot analysis of alpha-class glutathione S-transferases (alpha GST), enzymes known to inactivate the toxic AFB1-8,9-epoxide, showed a 2-fold increase in subunit levels in the DES-treated males, suggesting that the detoxifying activity of the cytosol may have been increased. To confirm this, in vitro tests were undertaken using butylated hydroxyanisole (BHA) induced mouse microsomes to activate [3H]AFB1 in the presence of treated cytosol and GSH. Analysis of metabolites by HPLC showed that DES-treated males formed 245% of the AFB-SG conjugate relative to vehicle controls. These results indicate that neonatal DES treatment resulted in long-term changes in basal alpha GST levels and suggest that these changes were responsible for lower levels of DNA adduction following adult exposure to AFB1.


Detection of mammalian carcinogens with an immunological DNA synthesis-inhibition test. There is a close relationship between genotoxicity, mutagenicity and carcinogenicity. But the controversy of which short-term test system best recognizes human carcinogens is still going on. Currently, the Salmonella gene mutation assay ('Ames test') is the most widely used test for the screening of mutagens. However, many in vitro tests hold unsatisfactory validity data, presumably because of the inability of present short-term tests to detect non-genotoxic carcinogens, which are increasingly being brought into focus in the discussions of genesis of cancer. One principle often neglected in this context is the property of genotoxic agents to inhibit replicative DNA synthesis in (proliferating) eukaryotic cells. We believe that this early response to DNA damage is important in the multistage process of carcinogenesis. Accordingly, we proposed that a DNA synthesis-inhibition test should be included in the test batteries for carcinogen screening. In this paper we report the development of an appropriate DNA synthesis-inhibition test based on immunological techniques.


Cytochrome P4501A induction in tissues, including olfactory epithelium, of topminnows (Poeciliopsis spp.) by waterborne benzo[a]pyrene. Topminnows of the genus Poeciliopsis are susceptible to hepatocarcinogenesis by waterborne exposure to procarcinogenic polycyclic aromatic hydrocarbons (PAH). We examined induction of cytochrome P4501A (CYP1A) in liver and other organs of the species P. monacha and P. lucida exposed to benzo[a]pyrene (B[a]P) in water (added in acetone carrier) at 1 mg/l for 48 and 90 h. Fish were fixed whole in formalin, and CYP1A was examined immunohistochemically in sagittal sections of whole animals by staining with monoclonal antibody 1-12-3, which recognizes a single cross-reacting CYP1A protein in Poeciliopsis liver microsomes. Fish exposed to B[a]P for 48 h showed moderate staining, and those exposed for 90 h showed strong specific staining in various epithelial cells in both species. These included hepatocytes, pancreatic cells, epithelial cells in gill, enterocytes of the gut, and kidney tubular epithelium. Endothelial cells in several organs, including gill pillar cells and endocardial cells in the heart, showed strong staining. Staining was stronger in P. monacha than in P. lucida. Untreated animals of both species showed mild staining of the same cells stained in B[a]P-treated fish. In P. monacha, carrier (acetone) elicited a moderate increase in staining in most cell types, including those of liver and gill; the basis for this acetone effect is not known. There was a very strong specific induction by B[a]P in olfactory epithelium and epidermal taste bud epithelium of P. monacha, the first demonstration of strong CYP1A induction in chemosensory epithelia exposed to inducer in a physiologically relevant way. This study clearly establishes that waterborne PAH can elicit induction of P4501A proteins in multiple cell types in many organs of fish, with some sites of induction (olfactory epithelium) possibly related directly to the route of exposure. The species differences in the induction response, with induction in liver and some other organs generally being greater in P. monacha than in P. lucida, could be related to previously recognized species differences in PAH toxicities in Poeciliopsis.


Epigenetic activation of Gi-2 protein, the product of a putative protooncogene, mediates tumor promotion in vitro. Promotion of 'initiated' JB6 epidermal cells to the tumor phenotype can be effected by 12-O-tetradecanoylphorbol-13-acetate treatment, by stimulation of epidermal growth factor (EGF) receptor activity with EGF or transforming growth factor alpha and by exposure to the isoquinoline derivative H7. When these cells were incubated with pertussis toxin (PTX), induction of anchorage-independent growth by all four promoting substances was suppressed. The inhibition is specific since cell proliferation is not affected, suggesting that activation of a Gi protein is essential for promotion of the epidermal cells. This interpretation is strongly supported by the observation that the wasp poison mastoparan, which is known to mimic receptor-mediated activation of certain Gi proteins, also promoted anchorage independence. Immunological data and partial amino acid sequence analysis of ADP-ribosyl alpha i isolated from PTX-treated JB6 cells indicate that a Gi-2 protein is a mediator to tumor promotion in this system. The inhibitory action of 4-bromophenacyl bromide may point to a coupling of the Gi protein to phospholipase A2. From our data we infer that promoters induce the tumor phenotype in 'initiated' JB6 epidermal cells by activating epigenetically the same Gi protein that in a number of adrenal and ovarian tumors appears to be persistently activated by mutational events.


A possible factor in genetic instability of cancer cells: stress-induced secreted proteins lead to decrease in replication fidelity. Ultraviolet irradiation triggers a response in mammalian cells known as the UV response. Part of the UV response forms the enhanced synthesis of various extracellular proteins able to transmit the response to non-irradiated cells. Because several cancer-prone syndromes with enhanced genetic instability also have an enhanced synthesis of the same set of proteins without prior stimulation it is possible that induction of these stress responsive proteins may be involved in the process of carcinogenesis and genetic instability. To test this hypothesis mouse T lymphoma cells, GRSL13, were treated with the conditioned medium of UV-induced cells under various experimental conditions. Overall the mutation rate is enhanced 1.8-fold (P < 0.01). However, the degree of enhancement is strongly influenced by culture conditions. UV-induced factors only lead to an enhanced mutation rate when cells, both for the production and response to those factors, originate from a similar cell density. In addition, it was found that fresh medium interferes with this response. To eliminate the hindrance of these factors on the effect of the conditioned medium on the mutation rate, serum-starved cells at high density were treated with serum-free medium derived from high-density UV-irradiated cultures. Using these conditions a 2.8-fold (P < 0.002) enhancement of the mutation rate was found. Fluctuation analysis indicated that the enhancement is 10-fold during the first five generations after treatment. UV-induced factors have also been found to induce cell growth, and the degree of induction was linearly correlated with the enhancement in mutation rate. These experiments are in agreement with the hypothesis that induction of stress responses leads to genetic instability.


Analysis of tobacco-specific N-nitrosamines in indoor air. A method was developed and applied for the assessment of tobacco-specific N-nitrosamines (TSNA) in indoor air polluted with tobacco smoke. Air samples were collected on Cambridge filters treated with 0.01 M potassium bisulfate, extracted with dichloromethane and enriched by column chromatography. The fraction containing the TSNA was concentrated and placed on a thermal desorption cartridge packed with Tenax GR. The sample was thermally desorbed and analyzed by capillary GC using a thermal energy analyzer. When the method was applied in a test laboratory in which one, two and four cigarettes were smoked during 30 min, linearity was observed. Field studies included sampling in bars, restaurants and trains. The concentration of N'-nitrosonornicotine (NNN) ranged from not detected to 23 pg/l, that of N'-nitrosoanatabine ranged from not detected to 9 pg/l, while 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) was detected in concentrations ranging from 1 to 29 pg/l. This means an exposure to NNN and NNK of 0.1-0.3 cigarette equivalents. Thus, non-smokers can be exposed to highly carcinogenic TSNA.


An in vitro pancreas acinar cell model for testing the modulating effects of caloric restriction and ageing on cellular proliferation and transformation. Pancreatic acinar cells were isolated for culture from a young (Y) and an old (O) Brown-Norway or Fischer 344 rat fed an ad libitum (AL) or calorically restricted (CR) diet. The cells were cultured and cellular growth rates were determined as a function of passage number. An overall increase in cellular growth rate and transformation frequency with age and/or AL diet relative to youth as well as a decrease with CR diet were concordant with reported responses in vivo. Transformation frequency was measured in Brown-Norway cells and followed the same pattern as the growth response: AL/O > AL/Y = CR/Y > CR/O. The cellular model is shown to fit the general multistage requirements of the carcinogenic process as well as general age and diet characteristics of pancreatic cancer. This pancreatic acinar cell age-diet approach may prove to be a valuable tool for determining mechanisms of exocrine pancreatic carcinogenesis as well as other disease states; it may also be of utility in in vitro gerontological nutritional and pharmacological studies since some of the age and diet determinants of biological effects appear to be segregable. Propensity of cells from an old and/or AL diet animal for faster growth and for cellular transformation are programmed into the cells by the time of their excision from the animal (as late as 14 months), indicating a heritable component in the model or a mechanism that is dependent upon elements that control gene expression.


In vivo and in vitro genotoxicity of several N-nitrosamines in extrahepatic tissues of the rat. Toxicological mechanisms involved in organotropism of tumor induction may include cell-specific metabolic activation of the carcinogen, in vivo distribution of active metabolites and persistance of induced DNA damage. In order to elucidate which factors are involved in the organotropic action of environmentally relevant N-nitrosamines, we have studied their genotoxic and cytotoxic effects within primary intact cells of lung and kidney. The end-points determined were cytotoxicity by trypan blue exclusion and DNA single-strand break (SSB) induction by alkaline filter elution. The assays were performed in vitro to determine organ-specific metabolic activation by incubating the cells with the test compounds. The results obtained with N-nitrosodimethylamine (NDMA), N-nitrosodiethylamine (NDEA), N-nitrosodiethanolamine (NDEIA), N-nitrosoethylvinylamine (NEVA), N-nitrosodibutylamine (NDBA), N-nitrosobutylbutanolamine (NBBOH), N-nitrosobutylcarboxypropylamine (BCPN), N-nitrosomethylbenzylamine (NMBzA) and N-nitrosodibenzylamine (NDBzA) indicate that several compounds may be activated to reactive metabolites by cells of kidney and lung, NDBzA revealing the highest degree of cytotoxicity. In contrast, genotoxicity in kidney cells was induced only by NBBOH and BCPN and at relative low levels. Primary lung cells could not be employed as indicators for genotoxic effects in vitro because the cell yield was not sufficient to perform the alkaline elution assay. To assess the distribution of NDMA in the whole rat and the persistence of the induced DNA damage in the two organs, further studies were carried out after oral application of 1, 2, 4, 10, 20, 32 and 40 mg/kg to the animals. Following 1 h exposure of rats to NDMA, the lowest effective genotoxic dose for lung and kidney was 2 mg NDMA/kg body wt. A plateau was achieved after a dose of 20 mg/kg in both organs. Furthermore, the persistence of DNA damage was studied in the lung. After 4 h exposure, DNA damage was still detectable at 32 mg NDMA/kg, but for the lower doses it was reduced nearly to control levels. After 16 h exposure the SSB rate in lung cells was reduced for all dose levels except for the highest dose of 40 mg/kg.


Pregnancy-induced antitumor cytotoxicity of T cell-rich fraction against mammary adenocarcinoma cells in rats. Our earlier observations indicated that splenocytes from parous rats have high cytotoxic activity against mammary tumor cells in vitro. It has also been observed that this cytotoxic activity of splenocytes from parous rats can be adoptively transferred to virgin rats of same age. The present investigation is an attempt to determine the cell type in spleens of parous rats that cause the cytotoxicity against mammary tumors. The spleens from both virgin and parous rats were removed aseptically and T and B cell-rich fractions were separated. 7,12-Dimethylbenz[a]anthracene-induced rat mammary tumor epithelial cells were used as targets and the T and B cell-rich fractions from either virgin or parous rats were used as effectors. The parous rats were divided into two groups according to the time period after parturition. Group I contained rats that were 5-13 days after delivery, while the rats in group II were 14 or more days post parturition. In vitro cytotoxicity was determined by incubating the tumor cells with spleen cells in the target/effector ratio of 1:3, 1:10 and 1:30 and using the standard 51Cr release assay. In all ratio groups of T cell-rich fractions particularly in group II, there was significantly higher cytotoxicity against mammary tumor cells. None of the B cell-rich fractions from parous rats were significantly more cytotoxic than those from virgin controls.