Occurrence and toxicokinetics of myosmine depending on smoking and nutrition

Myosmine, a minor tobacco alkaloid, occurs like the major tobacco alkaloids nicotine, nor-nicotine, anabasine and anatabine in small amounts in tobacco plants and tobacco smoke. Because of its low acute toxicity and the weak affinity to nicotinic receptors, no attention was given to myosmine for a long time. However, this changed with the discovery that myosmine occurs independently of nicotine in a great variety of staple foods, vegetables, fruits, nuts and dairy products and consequently in human milk, plasma and saliva. The possible health im¬pact of myosmine was further demonstrated by its conversion into reactive intermediates with carcinogenic potential. Due to its imine structure, myosmine is readily nitrosated and peroxi-dated. Nitrosation yields the tobacco-specific nitrosamine N'-nitrosonornicotine (NNN), a human carcinogen, which produces tumours in oesophagus, oral cavity and the respiratory tract of rodents. In larger quantities, the same reactive intermediates which are formed by me-tabolic activation of NNN and another tobacco-specific nitrosamine are generated by nitro-sation and peroxidation of myosmine. In biomonitoring studies the resulting DNA and protein adducts did not show the expected correlation with smoking status. Therefore, myosmine has been postulated to be an important additional source of these adducts.

In the first chapter, a detailed literature survey is given covering all aspects of occurrence, biosynthesis, toxicokinetics and -dynamics of myosmine in the context of other tobacco alkaloids and tobacco-specific nitrosamines.

In the experimental part, an analytical method was developed to determine myosmine, coti-nine and nicotine by gas chromatography/mass spectrometry (GC/MS) in different matrices in order to study the occurrence of myosmine in livestock as well as in humans. First, myosmine was determined in plasma of pigs in dependence of the feeding condition. Second, human sa-liva and toenails from smokers and nonsmokers were analyzed for short and long-term expo-sure to myosmine and cotinine. The nails were also analyzed for nicotine. Finally, myosmine was determined in multiple samples of saliva of eight test persons to study the kinetics of myosmine under controlled dietary conditions. The results can be summarized as follows:

• The analytical method has low detection limits for myosmine, nicotine and cotinine in toenails with 0.01, 0.02 and 0.035 ng/mg, respectively, and for myosmine and cotinine in plasma/saliva with 0.0012 and 0.05 ng/ml. The recovery is very high with 91-93% in plasma/saliva and 97–102% in toenails. The intraday precision is ≤ 8% for all analytes in toenails, whereas for plasma/saliva it is 18% with myosmine and 4% with cotinine. The analytical method has a high specificity by the use of deuterated internal standards.

• In the plasma of 12 fasting pigs, myosmine was traceable with only one exception. The concentration of myosmine was 0.067 ± 0.049 ng/ml. Within 1 - 2 hours after start of fee-ding the concentration of myosmine in the plasma of 13 pigs was 0.497 ± 0.166 ng/ml, a statistically significant 7-fold difference to the fasting pigs (p < 0.0001). The swill con-tained 124 ng myosmine/g wet weight.

• In toenails the concentrations of all analytes were significantly lower in 11 nonsmokers (0.021 ± 0.014 ng/mg) compared to 15 smokers (0.058 ± 0.052 ng/mg, p < 0.01). This 2.8-fold difference in myosmine concentrations between smokers and nonsmokers was clearly less than the 14-fold difference in nicotine concentrations (0.128 ± 0,008 versus 1.789 ± 0.964 ng/mg, p < 0.001). Cotinine was detectable only in toenails of smokers, 1.136 ± 0.843 ng/mg. Significant correlations exists between the concentrations of myosmine and nicotine (Spearman r = 0.67) as well as myosmine and cotinine (r = 0.63). A clearly better correlation (r = 0.83) was found between cotinine and nicotine values.

• In sal

The subject of the study was to investigate the aetiology of SARDS.

Following an initial literature review, patient data and clinical findings from 39 Patients were collected and analysed. One specific observation was that one third of all patients comprised Dachshunds and that 51.3 % of affected animals were female neutered. The average age of SARDS patients was found to be 8.8 years (range 3 months to 13 years). The ophthalmoscopic appearance of the fundus of SARDS patients was documented at different stages of the disease during serial examinations with the help of fundus photography. This revealed degenerative retinal changes which were found to progress linear following the onset of SARDS, finally resembling dogs with severe generalised progressive retinal degeneration.

In the second part of the study, the hypothesis that SARD is an autoimmune disease, was tested with the help of autoantibody screening. In a first step, serum from 24 SARDS patients and 14 normal controls were assessed for autoantibodies to the purified autoantigens S-Antigen and CRALBP with the help of a western blot. No difference in the incidence of autoantibodies could be found between SARDS patients and healthy controls while testing the well known autoantigens S-antigen and CRALBP.

In a second step, an attempt was made to identify new autoantigens by testing the entire retinal proteom as an autoantigenic source, again using Western blot techniques. Following the initial detection of a reaction against a specific protein found almost exclusively in SARDS patients, it was possible to identify this protein with the help of mass spectrometry (MALDI/TOF/TOF) as NSE. These findings were verified by the binding of IgG antibodies to purified NSE in 25% of the SARDS patients and 0% of the normal control dogs.

The results of this study suggest that an autoimmune aetiology of SARDS involving autoantibodies against NSE is possible. However, it is unclear whether these play a causative role in SARDS or whether they are the result of retinal destruction by another mechanism. Further investigations into a possible autoimmune aetiology of SARDS are therefore indicated and the role of NSE as an autoantigen must be further assessed.

Trotz verbesserter Therapiemöglichkeiten ist die Prognose beim Fibrosarkom der Katze noch immer ungünstig. In der vorliegenden Arbeit sollte die Verträglichkeit eines neuen Therapieansatzes zur Behandlung des felinen Fibrosarkoms untersucht werden. Verwendet wurde ein nonviraler Gentransfer mittels Magnetofektion, um eine Transfektion mit dem felinen Zytokingen GM-CSF zu erreichen. Ziel der durchgeführten Phase-I-Studie war die Festlegung einer maximalen tolerierten Dosis.

In die prospektive Dosis-Eskalations-Studie wurden Katzen, die definierte Einschlusskriterien erfüllten, aufgenommen. Die Steigerung der Dosis des Plasmids, das für feGM-CSF kodiert, erfolgte in vier festgelegten Schritten (50, 250, 750 und 1250 µg Plasmid). Jeweils vier Katzen wurden in eine Dosisgruppe aufgenommen. Die Plasmide wurden in wässriger Lösung in einem 1:1-Verhältnis mit magnetischen Nanopartikeln gemischt, die zur besseren Bindung an die DNA mit Polyethylenimin beschichtet waren. Die Plasmidlösung mit einem Volumen von 500 µl wurde intratumoral injiziert. Danach wurde durch Applikation eines Neodymium-Eisen-Bor-Magneten auf das Tumorgebiet ein Magnetfeld für die Dauer einer Stunde angelegt. Durch diese Magnetofektion wurde die Transfektion auf das Tumorgebiet beschränkt, sowie die Effektivität des Gentransfers verbessert. Das Behandlungsprotokoll umfasste zwei intratumorale Injektionen an den Tagen -14 und -7 sowie die großräumige en-bloc-Resektion des Fibrosarkoms an Tag 1. Eine Kontrollgruppe bestehend aus vier Katzen wurde ohne Zusatztherapie einer Operation unterzogen. Aus ethischen Gründen wurde dabei auf eine Verzögerung der chirurgischen Entfernung durch Placebo-Applikationen im Zeitraum von 14 Tagen verzichtet.

Eine Untersuchung auf mögliche auftretende Toxizitäten erfolgte an den Tagen -7, 0, 14, 45, 90 und 180. Zwei weitere Untersuchungen an den Tagen 270 und 360 dienten zur Kontrolle auf Rezidiv- und Metastasenbildung. Aufgetretene klinische oder hämatologische Toxizitäten wurden anhand eines speziellen Nebenwirkungskatalogs (VCOG-CTCAE-Tabelle) erfasst und in Korrelation zur durchgeführten Therapie gestellt, um über eine Zuordnung als Nebenwirkung entscheiden zu können. Ein statistischer Vergleich erfolgte für die Parameter Körpergewicht, Leukozytenzahl und das Differentialblutbild zwischen Kontrollkatzen, behandelten Katzen und den Katzen der verschiedenen Dosisgruppen. Sieben weitere Katzen wurden mit derselben Gentherapie mit humanem GM-CSF behandelt, bei denen der Expressionsachweis von huGM-CSF mittels ELISA in den Zellkulturüberständen der angezüchteten Tumore gelang.

In den ersten drei Dosisgruppen traten keine schwerwiegenden Nebenwirkungen auf. Da bei drei der vier Katzen der höchsten Dosis leichte Nebenwirkungen beobachtet wurden, wurden zwei weitere Katzen mit der höchsten Dosis behandelt. Dabei traten jedoch keine Toxizitäten auf, so dass die Dosis von 1250 µg für feGM-CSF kodierendes Plasmid als sichere, gut verträgliche Dosis für nachfolgende Phase-II-Studien festgelegt werden konnte. Auch die beobachteten Ergebnisse bezüglich Rezidivrate sind als sehr viel versprechend einzustufen.

The objective of the present study was to examine if heart murmurs in adult cattle can be objectively diagnosed using phonocardiography. The examinations were conducted using phonocardiograms (PCG) and required a scheme for evaluation. Twenty cattle older than two years of age that were hospitalised at the Clinic for Ruminants, Oberschleissheim, were used for the study. In all of them, heart murmurs were detected during auscultation.

The Meditron Analyzer 4.0 (Welch Allyn GmbH & Co KG, Jungingen) allowed the simultaneous recording of electrocardiogram (ECG) and PCG. The ECG using a thoracic lead served as a timeline for the PCG. In a pre-test (n = 20) so called threshold values were established which marked the division between the “background noise” (amplitudes between the heart sounds) and murmurs. These threshold values (for positive and negative range) were established for four different locations per side of the body (in the third and fourth intercostals spaces (ICS), dorsally and ventrally, re-spectively), as well as for the presentation of the PCG with use of a 50, 100 Hz filter, respectively. In the subsequent main study twenty animals were examined clinically and according to the results of auscultation assigned to the heart murmur grades (HM) 0-6 and categorised into groups: HM “faint” (HM grade 0-2), HM “moderately loud” (HM grade 3-4) and HM “loud” (HM grade 5-6). PCG were recorded on all eight locations mentioned above. The quality and number of acceptable PCGs of the right body side was very low, thus these recordings were not used in the analysis. For the PCG evaluation certain selected heart actions were presented in a larger scale and printed on graph paper. Systole and diastole were marked using the ECG and the threshold values were drawn in manually. In addition, systole and diastole were divided into 10 and 20 equal intervals for the 50 Hz and 100 Hz presentations, respectively. At these determined points the amplitudes were measured and recorded if they were above/below the threshold values or if no values could be measured at these points. For amplitudes that were outside the threshold values relative frequency at each point of measurement for each location and for 50 and 100 Hz presentations was calculated. Comparing the sum of the amplitudes outside the threshold values between the groups “faint” and “loud” significant differences were found for the location 1 (third ICS, dorsally, left side) in the systole at 50 Hz and for the loca-tion 2 (third ICS, ventrally, left side) in the systole at 100 Hz (p = 0.041 and p = 0.034).

A significant difference between the groups “faint” and “loud” was found for the loca-tion 1 in the systole at 50 Hz (p = 0.042) concerning the area under the curve using the amplitudes measured at these 10 and 20 points of measurement, respectively. In addition, the mean values of the amplitudes that were outside the threshold values were calculated. Comparing the groups “faint” and “loud” a significant difference was also found for the location 1 in the systole at 50 Hz in the positive range (p = 0.034) as well as for location 2 in the systole at 50 Hz in the negative range (p = 0.045).

Further investigations could confirm the tendencies found in the present study. Additional investigations with a larger number of animals could help to classify heart murmurs. Besides, they might help to increase the chances of obtaining usable measurements from the right side. Also, an algorithm for automatic evaluation of PCGs based on the data of the present study would be very helpful.