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Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

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Genetic monitoring detects an overlooked cryptic species and reveals the diversity and distribution of three invasive Rattus congeners in south Africa
Bastos, A.D.S. ; Nair, D. ; Taylor, P.J. ; Brettschneider, H. ; Kirsten, F. ; Mostert, E. ; Maltitz, E. ; Lamb, J.M. ; Hooft, W.F. van; Belmain, S.R. ; Contrafatto, G. ; Downs, S. ; Chimimba, C.T. - \ 2011
BMC Genetics 12 (2011). - ISSN 1471-2156 - 18 p.
mitochondrial-dna - native-range - black rats - prediction - rodentia - muridae - colonization - phylogenies - madagascar - radiation
Background: South Africa's long and extensive trade activity has ensured ample opportunities for exotic species introduction. Whereas the rich biodiversity of endemic southern African fauna has been the focus of many studies, invasive vertebrates are generally overlooked despite potential impacts on biodiversity, health and agriculture. Genetic monitoring of commensal rodents in South Africa which uncovered the presence of Rattus tanezumi, a South-East Asian endemic not previously known to occur in Africa, provided the impetus for expanded studies on all invasive Rattus species present.Results: To this end, intensified sampling at 28 South African localities and at one site in Swaziland, identified 149 Rattus specimens. Cytochrome b gene sequencing revealed the presence of two R. tanezumi, seven Rattus rattus and five Rattus norvegicus haplotypes in south Africa. Phylogenetic results were consistent with a single, recent R. tanezumi introduction and indicated that R. norvegicus and R. rattus probably became established following at least two and three independent introductions, respectively. Intra- and inter-specific diversity was highest in informal human settlements, with all three species occurring at a single metropolitan township site. Rattus norvegicus and R. rattus each occurred sympatrically with Rattus tanezumi at one and five sites, respectively. Karyotyping of selected R. rattus and R. tanezumi individuals identified diploid numbers consistent with those reported previously for these cryptic species. Ordination of bioclimatic variables and MaxEnt ecological niche modelling confirmed that the bioclimatic niche occupied by R. tanezumi in south Africa was distinct from that occupied in its naturalised range in south-east Asia suggesting that factors other than climate may influence the distribution of this species.Conclusions: This study has highlighted the value of genetic typing for detecting cryptic invasive species, providing historical insights into introductions and for directing future sampling. The apparent ease with which a cryptic species can become established signals the need for broader implementation of genetic monitoring programmes. In addition to providing baseline data and potentially identifying high-risk introduction routes, the predictive power of ecological niche modelling is enhanced when species records are genetically verified.
Bodemverontreiniging in de Biesbosch en doorvergiftiging naar kleine zoogdieren
Bosveld, A.T.C. ; Bie, P.A.F. de; Hamers, T. - \ 2003
Wageningen : Alterra (Alterra-rapport 654) - 64
bodemverontreiniging - zware metalen - polychloorbifenylen - vergiftiging - sorex araneus - muizen - muridae - fauna - histopathologie - enzymen - nederland - noord-brabant - zuid-holland - biesbosch - soil pollution - heavy metals - polychlorinated biphenyls - poisoning - mice - histopathology - enzymes - netherlands
In de Biesbosch worden bij overstromingen sedimenten afgezet op landbodem. Hiermee komen verontreinigingen zoals PAK's, PCB's en zware metalen beschikbaar voor de terrestrische voedselketen. Onderzocht is in welke concentraties deze verontreinigingen voorkomen in de landbodem en in hoeverre doorvergiftiging optreedt naar kleine zoogdieren. De voornamelijk planten en zaden etende woelmuis en de op bodemfauna (o.a. regenwormen) foeragerende bosspitsmuis zijn onderzocht. Dieren uit regelmatig overstroomde gebieden blijken hogere concentraties verontreinigingen in hun lichaam te hebben en vertonen duidelijke effecten op cytochroom P450 enzymfuncties (o.a. EROD). Daarnaast treden bij deze dieren in sommige gevallen ook effecten op in verschillende organen (lever, nier, gonaden) of op het lichaamsgewicht.
De noordse woelmuis in Noord-Holland midden; heden en toekomst
Nijhof, B.S.J. ; Apeldoorn, R.C. van - \ 2001
Wageningen : Alterra (Alterra-rapport 576) - 50
microtus oeconomus - populatie-ecologie - verspreiding - conservering - habitats - vegetatie - muridae - nederland - netwerken - habitatfragmentatie - fauna - knaagdieren - landschapsecologie - woelmuis - zoogdieren - Noord-Holland - population ecology - dispersal - conservation - vegetation - netherlands - networks - habitat fragmentation
Dit rapport beschrijft de verspreiding van de noordse woelmuis (Microtus oeconomus) in de provincie Noord-Holland. De relatie tussen voorkomen/verspreiding en vegetatie wordt beschreven. De analyse van de duurzaamheid van de huidige situatie wordt vergeleken met de duurzaamheid van de toekomstige situatie wanneer de provinciale ecologische hoofdstructuur uitgevoerd is. Knelpunten worden gesignaleerd en aanbevelingen gedaan om de duurzame instandhouding van de noordse woelmuis in Noord-Holland te garanderen.
De noordse woelmuis in Fryslân; naar een duurzame instandhouding
Nieuwenhuizen, W. ; Haye, M.J.J. La; Mertens, F. - \ 2000
Wageningen : Alterra (Alterra-rapport 149) - 52
microtus oeconomus - microtus agrestis - zoögeografie - distributie - habitats - inundatie - conservering - muridae - nederland - friesland - zoogeography - distribution - flooding - conservation - netherlands
Dit rapport beschrijft de verspreiding van de noordse woelmuis (Microtus oeconomus) in Friesland. Het voorkomen wordt besproken in relatie tot knelpunten en er worden aanbevelingen gedaan om te komen tot een duurzame instandhouding.
Om het behoud van de noordse woelmuis? Feiten en veronderstellingen
Apeldoorn, R.C. van - \ 2000
Zoogdier 11 (2000)2. - ISSN 0925-1006 - p. 24 - 28.
microtus oeconomus - muridae - muizen - bedreigde soorten - bescherming - conservering - habitats - territorium - milieu - populatie-ecologie - dierecologie - concurrentie tussen dieren - dieren - introductie - geïntroduceerde soorten - vrijgeven - genetische variatie - genetische diversiteit - genetische erosie - mice - endangered species - protection - conservation - territory - environment - population ecology - animal ecology - animal competition - animals - introduction - introduced species - release - genetic variation - genetic diversity - genetic erosion
Het belang van verschillende factoren die een rol spelen bij het behoud van de noordse woelmuis (Microtus oeconomus) in Nederland: habitatfragmentatie (versnippering); concurrentie met andere woelmuizen; genetische variatie en verarming (lokaal en regionaal). Aan de voorwaarden voor herintroductie is niet voldaan en er is meer onderzoek nodig
Dark-bellied Brent Geese Branta bernicla bernicla forego breeding when Arctic Foxes Alopex lagopus are present during nest initiation
Spaans, B. ; Blijleven, H.J. ; Popov, I.U. ; Rykhlikova, M.E. ; Ebbinge, B.S. - \ 1998
Ardea 86 (1998)1. - ISSN 0373-2266 - p. 11 - 20.
nesten - anser - ganzen - predatie - canidae - muridae - muizen - ratten - aziatisch rusland - stroomgebieden - nests - geese - predation - mice - rats - russian far east - watersheds
In an area north of the Pyasina delta in Taimyr (Russia), nest distribution, nest initiation and breeding success of Brent Geese Branta bernicla bernicla were studied in six successive summer seasons from 1990-1995 in relation to lemming and Arctic Fox Alopex lagopus abundance. Lemming abundance conformed to the well-known three-year cycle with peaks in 1991 and 1994. Wandering Arctic Foxes were numerous in 1992, one of the two years following a lemming peak. This was the only year in which foxes visited the small offshore island where Brent Geese used to nest. Although Brent Geese arrived in time that year, the majority did not even start to breed and disappeared. Thus the actual mechanism causing failure in 1992 was disturbance rather than predation and Brent Geese appeared to be able to forego breeding at the very last moment. In the unexpected absence of foxes in the second predator year 1995, Brent Geese incubated successfully on the small islands in our study area. However, they failed to raise their goslings as these were all predated, not by foxes but probably by gulls.
Non-homologous chromosome synapsis during mouse meiosis : consequences for male fertility and survival of progeny
Peters, A.H.F.M. - \ 1997
Agricultural University. Promotor(en): C. Heyting; P. de Boer. - S.l. : Peters - ISBN 9789054857761 - 182
muridae - muizen - meiose - geslachtelijke voortplanting - parthenogenese - polyembryologie - vruchtbaarheid - overleving - levensvatbaarheid - interacties - milieu - uitsterven - stofverplaatsing - chromosoomtranslocatie - chromosomen - cytologie - histologie - muridae - mice - meiosis - sexual reproduction - parthenogenesis - polyembryony - fertility - survival - viability - interactions - environment - extinction - translocation - chromosome translocation - chromosomes - cytology - histology
In the mouse, heterozygosity for several reciprocal and Robertsonian translocations is associated with impairment of chromosome synapsis and suppression of crossover formation in segments near the points of exchange during prophase of meiosis. This thesis describes the analysis of the consequences of the occurrence of non-homologous synapsis and/or suppression of meiotic crossover formation over many successive generations for male fertility and viability of the progeny.

For studying chromosome synapsis, we modified a drying down technique which results in high yields of nuclei of all first meiotic prophase stages in both male and female from only small amounts of tissue (chapter 2). Preparations are suitable for synaptonemal complex (SC) analysis by normal light and electron microscopy (chapters 2, 3 and 7), for fluorescence immunocytochemistry and in situ hybridization (chapters 2, 8).

In the study presented in chapter 3, we analysed the variation in male fertility of mice double heterozygous for two near identical reciprocal translocations T(1;13)70H and T(1;13)1Wa in relation to the synaptic behaviour of two differently sized heteromorphic bivalents during meiotic prophase. Male fertility rises when non-homologous synapsis in the small 1 13heteromorphic bivalent, leading to a "symmetrical" SC, is more frequent at the initial prophase stages. Based on the data presented, we favour the "unsaturated pairing site" model as the primary cause for male sterility.

In T70H/T1Wa females not all heterologous synapsis within the small heteromorphic bivalent is effectuated during the early stages of meiosis; some is achieved lateron by the mechanism of "synaptic adjustment" (chapter 3). Each heteromorphic bivalent contains a copy of the chromosome 1 region between the T70H and T1Wa breakpoints which is about 10 cM in size (Δ1 segment). Although axial elements representing these Δ1 segments are seen to approach each other during early meiotic prophase stages, they never successfully constitute a synaptonemal complex in either sex (chapter 3). This agrees with the fact that in earlier cytogenetic studies quadrivalents were never seen at both male and female diakinesismetaphase 1.

In chapter 7, we demonstrate that male fertility of the T70H/T1Wa mice is not only determined by the chromosomal constitution of the carrier but is additionally influenced by the pairing or synaptic history in previous meioses of especially the T70H and T1Wa short translocation chromosomes. Fertility of T70H/T1Wa males is more impaired after one or more successive transmissions of the T1Wa translocation chromosomes through a heteromorphic bivalent configuration, irrespective of the sex of the transmitting parent.

Furthermore, we show that the introduction of the Robertsonian translocation Rb(l1.13)4Bnr into the T70H/T1Wa karyotype restores fertility of double heterozygous males by stimulating non-homologous synapsis of the small heteromorphic bivalent. We speculate that this Rb4Bnr effect is mediated by a prolongation of the early stages of meiotic prophase I.

Successive female transmissions of the T1Wa translocation chromosomes in the presence of Rb4Bnr inititially resulted in an increase of the capacity for early meiotic nonhomologous synapsis within the small heteromorphic bivalent, leading to a restoration of fertility for the majority of carriers. Subsequently, a decrease of the capacity of the small heteromorphic bivalent to fully synapse was noticed, although a higher than original (F1) background level of male fertility remained.

These variations in male fertility are most likely based on epigenetic variance, reflected as the capacity to engage into non-homologous synapsis early in male meiosis leading to a "symmetrical" SC, despite the different amounts of chromatin to accommodate.

In chapter 4, the localization of several microsatellite markers and single copy genes relative to the T70H and T1Wa breakpoints, using quantitative PCR, quantitative Southern blotting and in situ hybridization, is described.

In chapter 5, we investigated the level of suppression of meiotic recombination and impairment of chromosome synapsis in T70H heterozygotes in relation to the viability of the progeny. For T70H/+ females, the introgression of the D1Mit4, D1Mit20 and D1Mit122 microsatellite marker alleles positioned distal of the T70H breakpoint on the normal chromosome 1 into the 13 1T70H long translocation chromosome was suppressed in a distance dependent manner. This effect was more pronounced in T70H/+ females, additionally homozygous for Rb4Bnr. The delay in introgression was paralleled by a reduction of the frequency and extent of non-homologous synapsis in segments near the T70H breakpoints of the pachytene translocation multivalents in T70H/+ and Rb4BnT70H/Rb4Bnr+ males. The extend of non-homologous synapsis around the centre of the synaptic cross configuration in these males correlated with fluctuations in prenatal viability of segregating translocation homozygotes in crosses between (Rb4Bnr)T70H homozygous males and heterozygous females when meiotic drive at the female second meiotic division is excluded. The reduction in viability is explained by the gain of mutations resulting from incorrect processing of recombination intermediates which is due to non-homologous synapsis around the translocation breakpoints.

In chapter 6, we analysed the consequences of the absence of crossing over for regions between the T70H and T1Wa breakpoints (Δ1 and Δ13 segments) of the Rb4BnrT1Wa translocation chromosomes, which have been transmitted for over 20 generations via heteromorphic bivalents in Rb4BnrT70H/Rb4BnrT1Wa females. Survival of heterozygous and homozygous carriers for these segments was taken as the phenotypic endpoint. The viability of progeny of crosses between Rb4BnrT70H homozygous males and Rb4BnrT70H/Rb4BnrT1Wa females, of which the latter principally produce 4 types of gametes, was estimated using a haplotype analysis of microsatellites in the Δ1 segment for genotyping (see chapter 4). We observed no differences in the pre- and postnatal survival rates of the double heterozygous and 13 1H, 13 1H, 1 13Wa 1 13H "duplication" progeny in which the Δ1 and Δ13 segments of the T1Wa translocation chromosomes had either no, an onegeneration or a multi-generation history of non-homologous synapsis in heteromorphic bivalents during previous female meioses. In addition, intercrossing of Rb4BnrT70H/Rb4BnrT1Wa double heterozygotes after genetic isolation of these Δ1 and Δ13 segments for 20 to 22 generations, showed that the viability of the Rb4BnrT1Wa homozygotes was not different from the Rb4BnrT70H homozygous and Rb4BnrT70H/Rb4BnrT1Wa karyotypes generated by this cross. Thus, exclusion of the Δ1 and Δ13 segments from meiotic crossing over within non-homologous synapsed heteromorphic bivalents during 20 to 25 successive generations does not result in an accumulation of recessive lethal mutations or an increased susceptibility for gaining dominant lethal mutations.

For the D1Mit122 microsatellite used in offspring haplotyping a higher mutation frequency was observed after transmission through a double heterozygous than after transmission through a T70H homozygous karyotype (chapter 6). On the basis of the identity of the mutations, the ectopic pairing of the St2 gene copies (containing D1Mit122) during meiosis of T70H/T1Wa males (chapter 8) and the observation of ectopic homologous contacts of the Δ1 segments during the zygotene stage without SC formation (chapter 3), we speculate that these mutations are the result of ectopic homologous gene conversion events most likely occurring in the absence of a synaptonemal complex.

The crossover suppressive influence of the Rb translocation on the Δ1 segment (chapter 5) enabled us to analyze the effects of introgression of genetic material from the Swiss +/+ stock into the translocation karyotypes. Introgression of "new" genetic material correlated with an increase in littersize of Rb4BnrT70H homozygotes (chapter 5), an improvement of the life expectancy of Δ1 duplication offspring from double heterozygous mothers (chapter 6) and a clear improvement of male fertility in double heterozygous and T70H homozygous males also carrying Rb4Bnr (chapter 7). These pleiotrophic findings are discussed in chapter 8 in terms of genetic versus epigenetic mechanisms of inheritance.

Finally, when T1Wa was backcrossed for many generations to the Rb4BnrT70H/Rb4BnrT70H karyotype, essentially precluding genetic recombination in the Δ1 and Δ13 segments, or when T1Wa was combined with Rb4Bnr after many successive transmissions via alternating T1Wa heterozygotes and homozygotes, stable Rb4BnrT1Wa homozygous lines could not be bred (chapter 8). Especially female reproductive performance decreases after repeated male and female homologous meiosis. As non-homologous synapsis in the centre of the synaptic cross configuration in T1Wa/+ males is common too (unpublished results), more work into the genetic stability of chromosome segments, that have a history of hindered homologous interaction, is indicated (chapter 8).

Changing gap junctional intercellular communication in mouse epidermal cells during tumorigenesis : a study on underlying processes
Jansen, L.A.M. - \ 1996
Agricultural University. Promotor(en): J.H. Koeman; W.M.F. Jongen; G.J. de Vrije. - S.l. : S.n. - ISBN 9789054855798 - 128
neoplasma's - muridae - muizen - signaaltransductie - oncologie - celinteracties - neoplasms - muridae - mice - signal transduction - oncology - cell interactions
Gap junctional intercellular communication (GJIQ plays an important role in the differentiation and growth of cells. Increasing evidence also suggests a role for inhibition of GJIC in the promotion phase of tumor formation. How the level of GJIC is regulated in normal cells, and if this regulation is changed during the process of tumor formation is however not clearly known yet.

In the chapters 3 and 4 the studies on the regulation of GJIC by (extracellular and intracellular) Ca 2+- and cAMP-dependent processes are described for a cell line consisting of initiated cells (3PC), and a carcinoma derived cell line (CA3/7). From these studies it can be concluded that differences exists between the regulation of GJIC in cells representing different stages in the process of tumor formation, and that the short-term regulation of GJIC by intracellular Ca 2+(Ca2+i) or by cAMP are different routes of regulation. Furthermore it can be concluded that the presence of cell adhesion molecule E-cadherin on the plasma membrane is a prerequested for a high GJIC level, but that E-cadherin is not involved in the short term regulation of GJIC by Ca2+ior cAMP.

The observed differences between 3PC cells and CA3/7 cells in the regulation of GJIC by intracellular signals suggest that during the process of tumor formation changes have occured in the Ca2+i- or calmodulin-dependent regulation of GJIC. These differences may be the result of differences in intracellular concentrations of regulating molecules (such as Ca2+ior CaM) or differences in the sensitivity of enzymes which are dependent of Ca2+ior CaM. These differences could result in a blocked intercellular communication between cell types representing different stages of tumor formation. For instance, if normal cells with a high GJIC level would come in contact with preneoplastic cells with different intracellular concentrations of calcium, a diffusion of calcium would appear at the moment intercellular gap junctions become functional between the two cell types. This diffusion of calcium will then lead to changed concentrations in the intracellular gap junction regions, which could result in mechanisms closing the gap junction to preserve the cellular homeostasis. Together this might lead to a quickly blocked GJIC between the normal and the preneoplastic cells, which could have a high homoloques GJIC level, as was shown between transformed and non-transformed Balb/c 3T3 cells.

Agents which decrease the level of GJIC (including tumor promoters) can play a role in the promotion phase of the process of tumor formation. Several test systems (based on the inhibition of GJIQ for the detection of agents with GJIC inhibiting capacity exist. In these assays however, the target cells for tumor promoters in the process of tumor formation (i.e. initiated cells) are not used. From the work presented in chapter 5 it can be concluded that a cell line consisting of initiated mouse epidermal cells (3PC) is a good model to detect agents with GJIC-inhibiting capacity, and that these cells are more sensitive for inhibition of GJIC compared to carcinomaderived cells. The sensitivity of primary keratinocytes compared to 3PC cells was varying and dependent on the agent used.

To determine if the differences between the cell types used for the detection of inhibition of GJIC (chapter 5) are attributable to differences in mechanisms which are thought to play a role in the regulation of GJIC (i.e. connexin amount, connexin phosphorylation, connexin location in the cell, E-cadherin amount, and E-cadherin location in the cell), we studied the effects of tumor promoters on these parameters in the different cell types (chapters 6 and 7). Because calcium plays a role in the regulation of GJIC (chapter 3), we also studied the effect of tumor promoters on Ca2+iand the role of Ca2+eon these effects (chapter 8). From these studies, the following conclusions can be drawn: 1) The mechanisms involved in the inhibition of GJIC by tumor promoters are agent- and cell type-dependent; 2) The observed differences in the susceptibility of cells for the inhibition of GJIC by tumor promoters can not be associated with effects of the studied agents on one of the studied parameters; and 3) tumor promoters can change [Ca 2+] i , but these changes are not associated with inhibition of GJIC.

In addition to the results of the studies on the regulation of GJIC (chapters 3 and 4), the studies with tumor promoters showed that, in the mouse epidermal cells used in these studies, only a decreased immunostaining of Cx43 on the plasma membranes of cells can be related to a decreased GJIC level. However, it must be noted that specific amino acid analysis of Cx43 could finally demonstrate if a relationship between inhibition of GJIC and Cx43 phosphorylation exists et all.

The mechanisms regulating the transport of connexins to and from the membrane, as well as the mechanisms involved in the formation of connexons are yet unknown. Because several tumor promoters inhibited GJIC in addition to delocation of both Cx43 and E-cadherin, it should be interesting to study the mechanisms involved in the assembly of gap junction proteins or cell adhesion molecules in the plasma membrane. Such study could lead to a better understanding of the mechanisms involved in (chemical-induced) inhibition of GJIC. A study on the time-related changes in tumor promoter-induced Cx43 inummostaining and E-cadherin immunostaining could give more insight in the role of E-cadherin in the regulation of GJIC. For such a study, also cells transfected with E-cadherin or a connexin could be used.

Zomernesten van de hazelmuis in Zuid-Limburg; ecologie en verspreiding
Foppen, R. ; Verheggen, L. ; Erkenbosch, H. - \ 1995
Natuurhistorisch Maandblad 84 (1995)8. - ISSN 0028-1107 - p. 200 - 212.
dieren - milieu - habitats - muizen - muridae - ratten - territorium - zuid-limburg - animals - environment - mice - rats - territory
Gebiedsgericht en soortgericht beleid in moerassen; de noordse woelmuis als toets
Bergers, P.J.M. ; Apeldoorn, R.C. van - \ 1995
Wageningen : IBN-DLO - 40
bescherming - dieren - uitsterven - bedreigde soorten - conservering - natuurbescherming - recht - wetgeving - beleid - bedrijfsvoering - muridae - muizen - zwampen - moerassen - wetlands - nederland - microtus oeconomus - protection - animals - extinction - endangered species - conservation - nature conservation - law - legislation - policy - management - mice - swamps - marshes - netherlands
Eerste vondst van de rosse woelmuis Clethrionomys glareolus op Schouwen-Duiveland
Bergers, P.J.M. ; Bussink, H. - \ 1995
Lutra 38 (1995). - ISSN 0024-7634 - p. 60 - 61.
dieren - invasie - muizen - migratie - mortaliteit - muridae - populatiedichtheid - populatie-ecologie - populatiegroei - ratten - clethrionomys glareolus - zeeland - animals - invasion - mice - migration - mortality - population density - population ecology - population growth - rats
Effects of vitamin A and [beta]-carotene on respiratory tract carcinogenesis in hamsters : in vivo and in vitro studies
Wolterbeek, A.P.M. - \ 1995
Agricultural University. Promotor(en): J.H. Koeman; V.J. Feron; A.A.J.J.L. Rutten. - S.l. : Wolterbeek - ISBN 9789054853749 - 158
carcinoom - neoplasma's - ademhalingsziekten - retinol - carotenen - provitaminen - carotenoïden - muridae - muizen - ratten - carcinoma - neoplasms - respiratory diseases - retinol - carotenes - provitamins - carotenoids - muridae - mice - rats
Summary
Respiratory tract cancer is the leading cause of death by cancer in 'Western' countries. The greater part of lung cancers are caused by smoking. Furthermore, environmental air pollution and occupational exposure contribute to the high incidence of lung cancer. Because it seems to be an almost impossible task to eliminate exposure of man to all these factors, considerable effort has been focused on identifying naturally occurring or synthetic compounds which can prevent the formation of respiratory tract cancer. In this regard, (pro)vitamin A (vitamin A and β-carotene) have been shown very promising. In a large number of epidemiological and experimental studies it has been shown that (pro)vitamin A inhibits the formation of respiratory tract cancer. However, the results of these studies are not always consistent and some studies even showed that (pro)vitamin A increases the incidence of lung cancer. Although the effect of (pro)vitamin A on the formation of respiratory tract cancer has been studied extensively, the mechanisms by which (pro)vitamin A influences the process of respiratory tract carcinogenesis are still not fully understood. In the studies described in this thesis, using both an in vitro and an in vivo approach, the effects of vitamin A and β-carotene on various stages of the process of chemically-induced respiratory tract carcinogenesis were investigated (Figure 1). The emphasis was on the effects of vitamin A and β-carotene on benzo[a]pyrene (B[a]P)-induced DNA-adduct formation, DNA-repair activities, cell proliferation and histomorphological changes in the hamster respiratory tract epithelium. Furthermore, the relationships between DNA-adduct formation, DNA-repair activities, cell proliferation and the expression of the tumour suppressor gene p53 were investigated.


In vitro studies
In the first in vitro experiments, the formation and repair of B[a]P-DNA adducts in hamster and rat tracheal epithelial cells was studied (Chapters 3 and 4). It was shown that in vitro the main DNA adduct formed in hamster tracheal epithelial cells was the trans-addition product of deoxyguanosine and (+)-anti-7,8-dihydroxy-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE-dG). This is the same DNA adduct as formed in vivo in tracheal epithelial cells of hamsters intratracheally treated with B[a]P. Furthermore, it is the same adduct as has been frequently observed in human respiratory tract cells. In rat tracheal epithelial cells two major DNA adducts were found in vitro : the BPDE-dG adduct and an adduct that is probably derived from interaction of syn -BPDE and deoxyadeno- s-ine. Both the formation of B[a]P-DNA adducts and the B[a]P-induced DNA-repair activities in hamster tracheal epithelial cells were time- and concentration-dependent. In rat tracheal epithelial cells, the formation of B[a]P-DNA adducts was 10 times lower than in hamster tracheas. Furthermore, unlike in hamster tracheal epithelial cells, B[a]P did not induce DNA-repair activities in rat tracheal epithelial cells. In the studies described in Chapter 5, the effect of vitamin A and β-carotene on the formation and repair of B[a]P-DNA adducts in hamster tracheal epithelial cells was investigated. It was shown that both vitamin A and β-carotene slightly inhibited the formation of B[a]P-DNA adducts. In addition, vitamin A and β-carotene increased B[a]P-induced DNA-repair activities. This suggests that the observed decrease in B[a]P-DNA adducts is a positive effect of vitamin A and β-carotene, probably also partly caused by an increase in DNA-repair activities. The effect of vitamin A on DNA-adduct formation and DNA-repair activities depended on the concentration of B[a]P versus the concentration of vitamin A. At a low B[a]P concentration relative to the concentration of vitamin A the formation of B[a]P-DNA adducts was inhibited by vitamin A, whereas at a relatively high concentration of B[a]P the formation of DNA adducts was enhanced by vitamin A.

The role of B[a]P and vitamin A in cell proliferation in hamster tracheal epithelium in organ culture is described in Chapter 6. It was shown that the effects of B[a]P and vitamin A on cell proliferation strongly depended on the culture medium used; in tracheas cultured in Ham's F12 medium cell proliferation was decreased by B[a]P treatment compared to control tracheas, while cell proliferation in tracheas treated with vitamin A in combination with B[a]P was increased compared to tracheas treated with B[a]P alone. In tracheas cultured in CMRL-1066 medium, the effects of B[a]P and vitamin A on cell proliferation were opposite to those observed in tracheas cultured in Ham's F12 medium: cell proliferation in tracheas cultured in CMRL-1066 medium and treated with B[a]P was increased compared to control tracheas, while vitamin A decreased B[a]P-induced cell proliferation. To explain these opposite effects of B[a]P and vitamin A on cell proliferation, various medium components and growth factors were investigated. The concentration of CaCl 2. 2H 2 O revealed to be the most important factor: supplementation of CaCl 2. 2H 2 O to the Ham's F12 culture medium mimicted the effects of B[a]P and vitamin A on cell proliferation in CMRL-1066 medium. These results clearly indicate that Ca 2+is an important regulator of proliferation of hamster tracheal epithelial cells. Furthermore, the results of these experiments showed that the level of B[a]P-DNA adducts was inversely related to cell proliferation in tracheas cultured in Ham's F12 medium. Although these results suggest that the tumour suppressor gene p53 might be involved by inhibiting cell proliferation as a consequence of DNA damage, we were unable to show a direct relationship between the level of B[a]P-DNA adducts, cell proliferation and expression of the p53 tumour suppressor protein in hamster tracheal epithelium in organ culture.

In vivo studies
The most widely applied in vivo model to study the aetiology and pathogenesis of respiratory tract cancer in experimental animals is based on repeated intratracheal instillations of a saline suspension of fine crystalline B[a]P particles attached to ferric oxide as a carrier. Various aspects of this method are discussed in Chapter 2, showing that the dose of B[a]P and the size of the B[a]P particles are the most important variables influencing the tumour response. In a first in vivo experiment into the effect of vitamin A and β-carotene on B[a]P-induced (pre)neoplastic changes in the respiratory tract of hamsters, the response of the respiratory tract epithelium was too low. This might be due to an insuficiently high B[a]P dose, possibly in combination with a relatively insensitive strain of hamsters used. The low response hampered studying potential effects of vitamin A or P-carotene on the (pre)neoplastic response (Chapter 7). An interesting observation in this experiment was an exceptionally low mortality of hamsters fed a high-β-carotene diet. Although we were unable to establish the exact cause of death of hamsters not receiving β-carotene, the most conspicuous difference between hamsters in the high-β-carotene group and hamsters in other groups was a decrease in lipid peroxidation in the livers of hamsters in the former group. Probably, this effect was not only due to the high concentration of β-carotene in the diet, but was also related to a high dietary level of α-tocopherol and ascorbyl palmitate (also present in the β-carotene beads and used to protect β-carotene from oxidation). To obtain a higher tumour response, hamsters were treated in a second experiment with a higher total dose of B[a]P (Chapter 8 and 9). In this study, a clear relationship appeared to exist between the extent of B[a]P-DNA adduct formation, the induction of cell proliferation and the immunocytochemically detected expression of the p53 protein in hamster tracheal epithelial cells. However, in this experiment the formation of B[a]P-DNA adducts was not found to be affected by a high dietary level of β-carotene, probably due to the high B[a]P dose. Furthermore, β-carotene did not affect B[a]P-induced cell proliferation and expression of the p53 protein in tracheal epithelial cells. Chapter 9 describes the histomorphological aspects of this hamster study, using conventional histopathology and immunohistochemical techniques for the detection of various cytokeratins and glutathione S -transferase (GST)-isoenzyme Pi. From this study, it appeared that B[a]P influenced both the expression of cytokeratins and the expression of the GST-isoenzym Pi. However, in accordance with the results described in Chapter 8, β-carotene did not inhibit B[a]P-induced lesions in the respiratory tract epithelium of hamsters.

Concluding remarks
Finally, the studies described in this thesis allow the following conclusions:

- In vitro, vitamin A and β-carotene decrease slightly but consistently the formation of B[a]P-DNA adducts, probably due to an increase in DNA-repair activities. The effect of vitamin A on the formation of B[a]P-DNA adducts depends on the concentration of B[a]P versus the concentration of vitamin A.

- The effects of vitamin A and B[a]P on cell proliferation in hamster tracheal epithelial cells in organ culture strongly depend on the tissue-culture medium used, in particular on the concentration of Ca 2+in the medium. The effects of B[a]P and vitamin A on cell proliferation observed in tracheas cultured in CMRL-1066 medium are similar to the effects generally observed in vivo .

- The hamster tracheal organ culture model is very suitable to study the B[a]P-induced formation of DNA adducts and DNA-repair activities. Both the formation and repair of B[a]P-DNA adducts is dose and time dependent. Furthermore, the main adduct formed in vitro is similar to the adduct formed in vivo after intratracheal instillation of B[a]P, and moreover, this adduct is frequently observed in man.

- A high dietary dose of β-carotene, possibly in combination with a high level of et-tocopherol and ascorbyl palmitate, strongly increases the survival of hamsters.

- In tracheal epithelia] cells of hamsters treated intratracheally with B[a]P, a relationship between the level of B[a]P-DNA adducts, cell proliferation and p53 expression is observed.

- The effect of vitamin A on B[a]P-induced DNA-adduct formation and cell proliferation, as observed in the in vitro experiments, was not found in in vivo experiments, probably due to the high B[a]P dose applied.

- β-carotene did not affect the formation of (pre)neoplastic changes in the respiratory tract epithelium of hamsters intratracheally treated with B[a]P as evaluated by conventional histopathology, cytokeratin expression, and glutathione S-transferase isoenzyme Pi expression.

- Although intratracheal instillation of B[a]P to Syrian golden hamsters is one of the most widely applied models to study respiratory tract cancer in experimental animals, the tumour response is difficult to control due to a large number of variables affecting the response. The most important variables influencing the tumour response are the dose of B[a]P and the size of the B[a]P particles.

In conclusion, although the in vitro experiments described in this thesis show that vitamin A and β-carotene may influence the process of respiratory tract carcinogenesis, in vivo it was not possible to show a modulating effect of vitamin A and β-carotene on B[a]P-induced respiratory tract cancer in hamsters. To explain the inconsistencies in the effect of vitamin A and β-carotene on respiratory tract cancer, further in-depth research should he focused on the molecular mechanisms underlying this effect. The concentration of vitamin A and β-carotene, in particular the concentration of the active metabolite retinoic acid, in target cells should be measured in relation to the action of these molecules on the genomic level.

Mouse models in leukemia
Voncken, J.W. - \ 1995
Agricultural University. Promotor(en): R.W. Goldbach; J.H.C. Groffen. - S.l. : Voncken - ISBN 9789054854166 - 148
gammaretrovirus - virologie - moleculaire biologie - leukemie - muridae - muizen - modellen - onderzoek - gammaretrovirus - virology - molecular biology - leukaemia - muridae - mice - models - research

Human Philadelphia-positive leukemia results from a balanced chromosomal translocation, which fuses the BCR gene on chromosome 22 to the ABL proto-oncogene on chromosome 9. The understanding of Ph-positive leukemogenesis has advanced enormously over the last few decades. Although in vitro assay systems currently used, are not always relevant to human tumor biology, much can and has been learned from studies, employing cell cultures and overexpression of BCR/ABL oncogenes.

Another restriction in leukemia research is the availability of primary human tumor material for study. Moreover, such tissues often represent terminally advanced stages of tumorigenesis. Therefore, the importance of in vivo models to study Philadelphia-positive leukemia is manifold. A well defined transgenic mouse model allows for tumorigenesis to be studied from its earliest stages onward and factors and mechanisms that eventually contribute to malignant progression of the leukemic cells can be uncovered. Besides an 'unlimited' provision of tumor material for analysis, more importantly, the availability of a transgenic mouse model provides a means by which cancer treatment regimes can be tested. In addition, identification of cellular components and/or pathways that contribute to the onset or progression of leukemia may eventually lead to the discovery and development of new drugs.

In 1990, Heisterkamp and co-workers reported on a transgenic mouse model for Philadelphia-positive acute lymphoblastic leukemia (ALL). Since most of the transgenic animals of an earlier study had succumbed to leukemia, part of the aim of this thesis was to generate BCR/ABL P190 transgenic founder animals de novo and to derive a transgenic animal line(s) which was to be used for future studies. In order to better understand the animal model, leukemogenesis was studied in great detail in transgenic founder animals and their progeny. In the second chapter a cytogenetic study of the mouse model for acute lymphoblastic leukemia is presented. Karyotypic analysis of leukemic bone marrow of a significant number of mice shows, that leukemic cells undergo a clonal development and karyotype evolution toward a more aggressive tumor: a high frequency of aneuploidy is found in advanced leukemia, as occurs in human leukemia, with a preference for gain of chromosomes 10, 12, 14 and 17. These findings are corroborated by experiments that reveal a gain of malignancy of the cancer upon serial transplantation of leukemic bone marrow to irradiated recipient mice and by molecular analysis of lymphomas using immunoglobulin rearrangement as an indicator for tumor clonality. The results suggest that BCR/ABL has a destabilizing effect on the regulation of the proces of mitosis.

In the third chapter, a correlation is described between the transcriptional status of the BCR/ABL P190 transgene and the development of leukemia: methylation of particular sequences in BCR exon-1 in the transgene is closely coupled to transgene inactivation, providing additional evidence for a direct role of BCR/ABL in leukemogenesis. A biological dissection of the oncogenic specificity of BCR/ABL is presented in the fourth chapter. Using sensitive molecular biological techniques, it is shown that, although expression of the BCR/ABL transgene is detectable in every tissue, from very early on in mouse development, no other neoplasias than of hematopoietic origin are found. The results strongly suggest that the oncogenicity of BCR/ABL is restricted to nucleated blood cells, which is very likely a reflection of cellular functions of the BCR and or ABL gene in signal transduction specific to hematopoietic lineages. The observations would also explain why the Ph-chromosome, which one would expect to arise by chance in many proliferating tissues, is found only in blood cancers.

An analysis of transgenic mouse models for chronic myelogenous leukemia, using BCR/ABL P210 transgenes is presented in the fifth chapter. The clinical disease spectrum includes differentiated and undifferentiated T and B cell leukemias. The myeloid compartment is implicated only sporadically and rather late in the disease process. In some instances, the observed myelo-proliferation is a sequel to deregulation of cytokine production at advanced stages of leukemia. The course of P210 induced leukemia was acute rather than chronic, be it with an on average longer latency period than typical for ALL in BCR/ABL P190 mice. From these studies is was concluded that in the mouse, BCR/ABL P210 evokes a clinically different disease than BCR/ABL P190. Although no evidence for a chronic myeloproliferative disorder in the peripheral blood was found, an imbalance in myelopoiesis in the bone marrow suggests an effect of BCR/ABL P210 on primitive myeloid progenitors.

The sixth chapter summarizes an analysis of interferon-α(IFN-α) treatment of the BCR/ABL P190 transgenic mice. (IFN-α) is currently one of the most effective drugs in the treatment of CML. Recently, (IFN-α) was tried in the treatment of ALL. No effect of (IFN-α) on animal survival or disease pattern were noted when administered to the BCR/ABL P190 mice. The conclusion was reached that, at least in a transgenic setting, (IFN-α) does not interfere with BCR/ABL P190 mediated leukemia.

In order to study the normal cellular function of the BCR gene and to eventually assess its role in leukemogenesis, studies focussing on the mouse bcr gene function are presented in chapters 7 and 8. The seventh chapter describes the ablation of a functional mouse bcr gene by means of recently developed gene targeting techniques. One of two mouse bcr alleles was inactivated in a mouse embryonic stem cell line through gene interruption by insertional replacement vectors. Ibis genetically altered cell fine was then injected into developing mouse embryos. Through germline transmission of the mutated allele and subsequent breeding both bcr alleles were inactivated. Although bcr -null mutants are phenotypically normal, their neutrophils display impaired regulation of respiratory burst, which becomes apparent when these cells are activated in vivo: an overproduction of superoxide leads to significantly more oxidative tissue damage during experimental endotoxemia. The results connect Bcr in vivo with the regulation of superoxide production by the NADPH- oxidase system of leukocytes and suggest a link between the cell types affected by loss of Bcr function and the those involved in Ph -positive leukemia.

Additional information on biological processes that Bcr participates in, are described. in the eighths chapter. Notwithstanding its function in hematopoietic cells, the Bcr protein is normally found in high levels in brain. The expression pattern of bcr in rodent brain was examined by means of in situ hybridization and Northern analysis. Although not directly connected with leukemogenesis, a potentially interesting role for p160Bcr in the brain is discussed as its expression pattern appears to coincide with the functional organization of particularly highly specialized structures in the brain.

With the availability of well defined transgenic mouse models for BCR/ABL positive leukemia, an opportunity is created to study the nature of cellular interactions and processes that contribute to the onset and development of Ph -positive leukemia. Ultimately, such investigations are aimed at designing and testing effective therapeutic drugs to fight the disease.

Molmuis ondergraaft vegetatie-ontwikkeling.
Kooy, J. van der; Maanen, B. van - \ 1994
Zoogdier 5 (1994)2. - ISSN 0925-1006 - p. 3 - 7.
dieren - begrazing - herbivoren - muizen - muridae - plantensuccessie - ratten - verstoring - limburg - animals - grazing - herbivores - mice - plant succession - rats - disturbance
Prevention of vole damage on trees : a review of literature
Moraal, L.G. - \ 1993
Wageningen : IBN-DLO (IBN research report 93/7) - 14
bosschade - wilde dieren - muridae - muizen - ratten - bosbouw - fysische gewasbeschermingsmethoden - gewasbescherming - rodenticiden - overzichten - schadelijke dieren - mechanische bestrijding - forest damage - wild animals - mice - rats - forestry - physical control - plant protection - rodenticides - reviews - noxious animals - mechanical control
Zygote development after delayed fertilization : a cytological and genetical analysis of embryos of the mouse
Boerjan, M.L. - \ 1990
Agricultural University. Promotor(en): C. Heyting; P. de Boer. - S.l. : Boerjan - 90
muridae - muizen - geomyidae - eieren - ontwikkeling - eivorming - oögenese - dierlijke weefsels - beenweefsel - dieranatomie - muridae - mice - geomyidae - eggs - development - egg formation - oogenesis - animal tissues - bone tissue - animal anatomy

Chapter I gives (1) a brief review of morphological and molecular changes in mammalian oocytes initiated by fertilization, (2) a summary of published data on several aspects of post-ovulatory ageing in relation to embryonic development and (3) a description of methods to time ovulation and fertilization.

Precise timing of ovulation and fertilization was a prerequisite for the study of the consequences of delayed fertilization for embryonic development.
Chapter II describes the methods of ovulation induction and artificial insemination. We induced ovulation with injections of luteinizing hormone releasing hormone (LHRH) at pro-oestrus of the oestrus cycle. The number of oocytes that ovulated after LHRH administration was similar to that after spontaneous ovulation. Although LHRH was administered 8-12 hrs before the expected endogenous luteinizing hormone (LH) surge, we found no significant effect of LHRH induced ovulation on embryonic mortality. This method of ovulation induction, supplemented with artificial insemination to achieve in vivo fertilization, provided a tool to study aspects of early embryonic development after delayed fertilization.

Investigations were carried out to determine in detail the timing of the distinct morphological changes triggered by fertilization of unaged oocytes and oocytes aged post-ovulation for 12 hrs. Sperm penetration was shown to be accelerated by 1 hr 30 min after delayed insemination compared with sperm penetration in unaged oocytes. The rate of progression to the first cleavage division was also influenced by the post-ovulation age of mouse oocytes prior to fertilization: penetrated aged oocytes needed less time (1 hr 30 min) to reach the 2-cell stage than zygotes from unaged oocytes. This observation was confirmed by experiments carried out later: the percentages of 3- and 4-cell embryos from aged and unaged oocytes, collected 36 hrs after insemination, were 14% and 7% respectively (Chapter IV).

Fertilization activates, among other things, a cascade reaction of protein synthetic alterations.
Chapter III deals with protein synthetic activity in unaged and aged LHRH induced oocytes, in unaged superovulated oocytes and in zygotes derived from these types of oocytes. Polypeptides with a relative molecular weight of 35 kDa were predominantly synthesized by LHRH induced and superovulated secondary oocytes and zygotes from these oocytes. This study of patterns of 35 kDa proteins synthesized by zygotes from aged and unaged LHRH induced oocytes revealed that fertilization dependent protein synthetic changes of the 35 kDa protein complex were advanced in zygotes from aged oocytes with reference to pronuclear development.
A fraction (16.7%) of morphologically normal zygotes from unaged superovulated oocytes did not synthesize the 35 kDa protein complex at all.

It was of interest to learn more about the in vitro developmental capacity of embryos from aged oocytes.
In Chapter IV the results of these investigations are arranged and discussed. One-cell and late 2-cell embryos from aged and unaged oocytes were cultured in the presence and absence of DNA damage or DNA-damaging agents for different periods of time. On the one hand we studied the progression to metaphase of the first cleavage division in zygotes from aged and unaged oocytes fertilized with X-irradiated sperm. On the other hand the in vitro developmental capacity of late 2-cell embryos was evaluated in presence and absence of the thymidine analogue 5-Bromode-oxyuridine (BrdU).

Post-ovulatory ageing had an effect on the morphology of male as well as female pronuclear chromosomes of the first cleavage metaphase. We also found a detrimental effect of fertilization with X-irradiated spermatozoa on the morphology of male and female pronuclear chromosomes. This effect was particularly observed in male pronuclear chromosomes of zygotes from aged oocytes. Furthermore, fertilization with X-irradiated spermatozoa led to an arrest at interphase in 27% and 7% of zygotes from aged and unaged oocytes respectively. This arrest was not shown after fertilization with sperm not irradiated with X-rays.

This experimental setup also enabled us to compare the amount of radiation induced chromosome damage in zygotes from aged and unaged oocytes. Zygotes from aged oocytes did not contain more chromosome damage than zygotes from unaged oocytes, when the visible chromosome mutations originating from the X-irradiated spermatozoa were analyzed at metaphase of the first cleavage division.
In Chapter IV we also have shown that zygotes from aged oocytes and unaged oocytes develop in vitro at similar rates from the late 2-cell stage, collected at 36 hrs after insemination, to the 8-cell stage (24 hrs cultures). However, in vitro development of 2-cell embryos from aged oocytes collected 30 hrs after insemination and cultured for 66 hrs is impaired.
To determine the number of sister-chromatid exchanges, we cultured 2-cell embryos from aged and unaged oocytes, collected 36 hrs after insemination, in the presence of BrdU for 24 hrs. SCE levels were not significantly different between embryos from aged and unaged oocytes.
Cell proliferation of late 2-cell embryos from aged oocytes, collected 36 hrs post-insemination, from aged oocytes was clearly retarded and asynchronous during the 24 hrs culture period in the presence of 10-6 M BrdU.

In Chapter V a cytochemical method is described to determine in individual oocytes the distribution of the activity of SDH (succinate dehydrogenase), an enzyme which is located on the inner membrane of mitochondria. We showed that treatment of oocytes with the drug caffeine prior to cytochemical staining resulted in an intens staining of the cells by a formazan precipitate. We applied the cytochemical staining procedure to preovulatory oocytes of mice. In a maturation experiment in vitro we found that the location of formazan correlated well with the location of mitochondria in subsequent stages of maturation. Unfortunately, this cytochemical staining procedure could not be applied to ovulated and fertilized oocytes, since these cells acquired a poor morphology during the staining procedure and displayed high levels of non -dehydrogenase formazan production.

Cytokinetics and histogenesis of cultured hamster tracheal epithelium : effects of vitamin A and cigarette smoke condensate
Rutten, A.A.J.J.L. - \ 1988
Agricultural University. Promotor(en): J.H. Koeman; J.W.G.M. Wilmer. - S.l. : Rutten - 159
muizen - muridae - ratten - retinol - tabak roken - toxicologie - mice - muridae - rats - retinol - tobacco smoking - toxicology

The studies reported in this thesis primarily deal with the influence of vitamin A (all-trans retinol) and cigarette smoke condensate on cellular proliferation, and differentiation and intercellular communication in tracheal epithelium. The experiments were carried out with Syrian Golden hamster tracheas and primary tracheal epithelial cells maintained in serum-free, hormone-supplemented media. All-trans retinol was used to regulate cellular proliferation and differentiation, whereas cigarette smoke condensate was used to induce alterations.

This thesis is divided into three parts: General introduction (Chapters 1 and 2), Experiments (Chapters 3-8) and Conclusion (Chapters 9 and 10).

In Chapters 1 and 2 of the current knowledge of retinoids, tracheal epithelium, intermediate filaments, intercellular communication and in vitro model systems with tracheal epithelium are summarized.

In Chapters 3 and 4 the effects of all-trans retinol and cigarette smoke condensate on vitamin A-depleted hamster tracheal epithelium in organ culture are discussed with emphasis on cellular proliferation and differentiation.

Chapter 5 deals primarily with the role of proliferating basal cells in untreated tracheal epithelium, and in vitamin A-depleted tracheal epithelium treated with a physiological all-trans retinol concentration. The effects on tracheal epithelial cells were observed by means of monoclonal antibodies against various keratins. The information obtained in this Chapter was used to study the effects of cigarette smoke condensate and vitamin A depletion on intermediate filament expression patterns in tracheal epithelium (Chapter 6).

In Chapter 7 experimental evidence is given that ciliated tracheal epithelial cells can divide in hamster tracheal organ cultures treated with all-trans retinol or cigarette smoke condensate.

Chapter 8 deals with the role of vitamin A (all-trans retinol and retinoic acid) and cigarette smoke condensate on dye-coupled intercellular communication between primary hamster tracheal epithelial cells.

In Chapters 9 and 10 a summary of the results is given with concluding remarks in both English and Dutch.

Mechanistic and quantitative aspects of liver tumour promotion in mice
Ravenzwaay, B. van - \ 1988
Agricultural University. Promotor(en): J.H. Koeman, co-promotor(en): H.A. Tennekes. - S.l. : Van Ravenzwaay - 166
lever - leverkanker - muizen - muridae - neoplasma's - liver - liver cancer - mice - muridae - neoplasms
A variety of xenobiotic compounds is known to induce characteristic changes in the livers of laboratory animals. These changes include enlargement of the liver, usually as a result of cell enlargement (hypertrophy) or Increased cell replication (hyperplasia), induction of drugmetabolizing enzymes and proliferation of the smooth endoplasmic reticulum (SER). Such changes are usually not accompanied by evidence of liver damage and thus are reversible upon withdrawal and elimination of the compound. Consequently, most authors regard this phenomenon as an adaptive response of the organ to increased functional demands.

However, chronic exposure of various strains of mice to dieldrin, phenobarbitone, DDT and the α-, β- and γ-stereoisomers of hexachlorocyclohexane (HCH, also known as benzenehexachloride, MC) may lead to the development of liver tumours.

The tumorigenic effects of microsomal enzyme Inducers In mice may result from (A) a weak carcinogenic action of the xenobiotics themselves or (h) an enhancing (promoting) action of xenobiotics on a pre-existing oncogenic factor in mouse liver. The first objective of this study was to discriminate between these two possible types.

Druckrey and his associates have established both theoretically and experimentally the dose-response characteristics of chemical carcinogens:

D.T n= constant (1)

where D = daily dose, T = the median tumour Induction period and n = an exponent, always>1.

Since the mechanisms by which enhancers or promotors of carcinogenesis operate is quite different from the one used by carcinogens, it is be conceivable that promotors also exhibit different dose-response characteristics.

The dose-response characteristics of dieldrin-mediated enhancement of liver tumour formation in CF-1 mice were analysed, using existing tumour data from chronic feeding studies at six exposure levels of dieldrin (a model compound for microsomal enzyme induction). It was found that the dose- response relationship can be expressed as:

(d o + δx).t = constant (2)

where d o stands for the background dose equivalent required for the induction of spontaneous liver tumours, δx represents the actual dieldrin dose (ppm in the diet) and t the median tumour induction period in the respective treatment groups. It was also established that the dose
response characteristics of limited dieldrin exposures and those of de layed exposure were consistent with equation (2), which is a Druckrey relation where n = 1.

From these findings it is concluded that dieldrin interacts reversibly with its receptors, resulting in an acceleration of tumour formation (which is essentially Irreversible); dieldrin may thus be regarded as a tumour promotor. The validity of equation (2) for both chronic and limited dieldrin exposure Indicates that ( a ) the velocity of liver tumour development is proportional to the daily dose level (δx), ( b ) the total tumorigenic dose is constant across all doses, ( c ) the effects of dieldrin on the neoplastic process In mouse liver are essentially irreversible and cumulative, and ( d ) there is no evidence for a threshold level.

Tumour formation Is a dose- and time-dependent process. The induction of liver enlargement, microsomal enzyme systems and proliferation of the smooth endoplasmic reticulum by dieldrin are only dose-dependent. In contrast, polyploldization Is dose- and time-dependent. To establish a
possible link between microsomal enzyme induction, nuclear polyploidization and liver tumour formation, nuclear polyploidization in livers of CF-1 mice was studied at five different dieldrin dose levels from 1.85 months up to tumour development. Nuclear polyploidization, expressed in the proportion of octaploid (8c) nuclei, was found to be characterized by a linear increase with age in untreated control CF-1 mice. Dieldrin treatment induced a dose-dependent increase in the proportion of 8c-nuclei in the initial phases of treatment. In "steady-state" situations nuclear polyploidization (as expressed by the percentage of 8c-nuclei) was maintained on a dose-dependent, higher level, and the percentage was was observed to increase with age, the velocity of which was the same as in untreated controls. Tumour formation was found to be associated with a constant degree of nuclear polyploidization In all treatment groups Including controls. The observed quantitative link between nuclear polyploidization and tumour formation leads to the question whether or not a causal relationship between the two exists. Assuming that polyploidization reflects the ageing process, the data suggest that liver tumour formation Is Imminent at a constant biological age and that dieldrin could operate by advancing the biological age of CF-1 mouse liver.

Further support for this hypothesis was obtained from the determination of cytoplasmic alanine amino transferase (AAT) isoenzymes. The expression of the isoenzyme decreases with age In untreated control CF-1 mice. Dieldrin treatment was found to enhance (accelerate) this process in a dose-dependent manner.

Although the nature of the development of "spontaneous" liver tumours in CF-1 mice remains unknown, the decrease In the tetraplold(4c)-diplold (2c) ratio of liver nuclei, observed in the study of polyploidization, may be related to tumour formation. The decrease was observed in all
treatment groups, including controls, and its onset was dose-dependently advanced by dieldrin treatment, occurring approximately 4 months before the median liver tumour induction period in all cases. Two mechanisms are proposed that may explain the tumorigenic features of a decrease
in the 4c-2c ratio.
1. Tetraploid cells could be more sensitive to accumulative toxic stress. Thus, their turnover may be Increased. To replace one tetraploid cell a diploid cell has to divide twice; the loss of tetraploid cells would therefore result in a proliferative response of the diploid
population (resulting tumour formation).
2. A reduction in the 4c-2c ratio could be induced by the occurrence of amitotic nuclear divisions in the tetraploid cells. Evidence for this possibility was obtained from experiments with 3H-thymidine-labelled nuclei. Amitotic nuclear divisions could give rise to chromosomal
re-arrangements, resulting in the expression of the intrinsic neoplastic potential of CF-1 mouse liver.
Both hypotheses imply that the diploid population is the source of liver
tumours. The determination of nuclear polyploidization in liver tumours confirmed that these tumours originate from the diploid liver cell population.

Meiotic behaviour and spermatogenesis in male mice heterozygous for translocation types also occurring in man
Nijhoff, J.H. - \ 1981
Landbouwhogeschool Wageningen. Promotor(en): J. Sybenga, co-promotor(en): P. de Boer. - Wageningen : Nijhoff - 141
muridae - muizen - meiose - stofverplaatsing - chromosoomtranslocatie - spermatozoön - spermatogenese - sperma - muridae - mice - meiosis - translocation - chromosome translocation - spermatozoa - spermatogenesis - semen
In this thesis a start was made with meiotic observations of mouse translocation types - a Robertsonian translocation and a translocation between a metacentric and an acrocentric chromosome - which also occur in man. It is generally accepted that, when no chromosomal rearrangements are involved, man shows a higher level of non-disjunction than the mouse. When the meiotic behaviour of translocations in mouse and man would be more similar than that found for the autosomal bivalents (and sex bivalent) of these species Chapter 1 reviews the methodology to establish this difference mouse systems characterized by chromosome aberrations would become more attractive as models for the study of endogeneous and exogeneous factors on the meiotic process and extrapolation from mouse to man would be facilitated. As it appeared in these studies that not only the translocation complex but also the normal bivalents contribute to a significant extent to the non-disjunction rates in the heterozygote for a translocation of the meta/acro type, this material appeared even more favourable as an experimental substitute for man than was initially expected.
Meiosis in male mice, heterozygous for the Robertsonian translocation Rb(11.13)4Bnr, has been studied in an attempt to elucidate the mechanism(s) of non-disjunction for the trivalent formed at meiosis I by the Rb chromosome and the two acrocentric homologues 11 and 13 (Chapters 2 and 3). As an exogeneous factor of possible influence, the meiotic effects of two types of radiation, administered at relatively low doses 2 and 3 hours before prometaphase-metaphase II (probably during metaphase-anaphase I), were determined in Rb4Bnr/+-males (Chapter 3).
The segregational behaviour of the multivalent at anaphase I has been compared in T(1;11.13S),+/+,RBnr, structurally homozygous for the metacentric Rb(11.13)4Bnr chromosome whereby arm 13 of one of the two metacentrics is involved in a reciprocal translocation with the acrocentric chromosome 1, and T(I113)70H,+/+,Rb4Bnr (Chapter 4). The multivalents formed by the latter karyotype at meiosis I are highly comparable to those of the former one; for details see section III below.
The results obtained, and presented below, warrant a further investigation of these mouse meiotic systems as models for the situation in man as is for example indicated by the significant increase of normal bivalent non-disjunction found in the T(1;11.3S),+/+,Rb4Bnr and the T70H,+/+,Rb4Bnr karyotypes.
In the next sections the chapters on which the conclusions are based, have been indicated between parentheses.

I Indications for the relation between delay before and around anaphase I and non-disjunction in male Rb4Bnr Robertsonian translocation heterozygotes

- For Rb4Bnr/+ males, inter mouse variation was noted with respect to the duration of the meiotic prophase, i.e. the period "end of the premeiotic S phase-metaphase V'. This was concluded on the basis of the frequency of late diplotene/metaphase I cells with labelled chromosomes recorded autoradiographically in mice killed various time intervals after the i.p. administered injection with 3 H-thymidine (3).

- For Rb4Bnr/+ males, compared to their +/+ controls, a greater mean number of late diplotene/metaphase 1 cells was counted in the separate groups along the seminiferous epithelium, whereas the mean distance between the subsequent groups was smaller. As these data are in contradiction with the reduction by 58% of the epidydimal sperm count for Rb4Bnr/+ compared to +/+, the greater number was thought to be an accumulation effect due to delay during diplotene/metaphase I for at least a fraction of the cells. Implicitly a causal relation between delay and cell death before the spermatozoal stage is also suggested (2).

- The duration of the period "end meiosis I (metaphase I) - end meiosis II'' was approached autoradiographically for Rb4Bnr/+-males with the aid of cells showing a labelled Y chromosome only. The best assessment of the duration of this period was 3 hours or less. Inter mouse variation with respect to the duration of the period "end of the premeiotic S phase-metaphase ill (see above) prevented a more accurate estimate (3).

- Irradiation of Rb4Bnr/+ males for 14.5 minutes with a dose of 15.2 rad fast neutrons (mean energy 1.7 MeV) significantly
decreased the incidence of ancuploid secondary spermatocytes, scored 2 and 3 hours after application. This was explained by selective cell killing of those cells giving rise to aneuploid daughter cells after anaphase I, rather than by protection against non- disjunction by the irradiation dose, as follows: For Rb4Bnr/+ during the final period of the primary spermatocyte stage, cells show a continuous distribution with respect to the extent of delay. Cells with a strong delay will die off before the secondary spermatocyte stage, while the fraction showing only minor delay will survive (in the unirradiated situation) but with an increased risk of producing aneuploid daughter cells. The results suggest that, in terms of cell death, the victims of the 15.2 rad neutron irradiation predominantly have to be searched in the last category. A causal relation between delay and ensuing non-disjunction at anaphase I, and between delay and the neutron irradiation effect respectively, is thus suggested but the basis for these interactions remains unknown (3).

II Irradiation induced damage during meiosis in male Rb4Bnr Robertsonian translocation heterozygotes

- After irradiation of Rb4Bnr/+ male mice for 14.5 minutes with either a dose of 15.2 rad neutrons or 60 rad X rays, only neutrons affected, viz. significantly decreased (see above), the incidence of aneuploid secondary spermatocytes estimated 2 and 3 hours after irradiation. The reason for this difference remained unknown and could not be explained on the basis of a difference in the levels of radiation induced damage. The two radiation types induced comparable levels of chromosome damage, i.e. numbers of breaks, fragments and deletions per cell, when compared in either late diplotene/metaphase I or metaphase II cells. For these parameters the neutrons: X ray RBE ratios estimated were 5.4 for meiosis 1 and 3.3 for meiosis II cells (3).

- After an irradiation-fixation interval of 2 to 3 hours, the chromosome damage (i.e. numbers of breaks, fragments and deletions) induced by either 15.2 rad neutrons or 60 rad X rays was
5-10 times higher when scored in metaphase II than in diakinesis/ metaphase I cells. At the moment of irradiation the two categories of cells were, most probably, at prometaphase/metaphase 1 and late diplotene/early diakinesis, respectively. The difference in chromosome damage was argued to be merely the consequence of differences in chromosomal processes taking place during the irradiation-fixation interval and not a reflection of a difference in radiation sensitivity between the two meiosis I stages (3).

III The meiotic behaviour in T(1;11.13S),+/+,Rb4Bnr and T70H,+/+,Rb4Bnr male mice, also with reference to the behaviour of the normal bivalents

The T(1;11.13S),+/+,Rb4Bnr karyotype includes the metacentric Rb(11.13)4Bnr chromosome in homozygous condition whereby the 13 arm of one of the metacentrics is involved in a reciprocal translocation with the acrocentric chromosome 1. The T70H,+/+,Rb4Bnr karyotype is double heterozygous for the T(1;13)7OH reciprocal translocation and the metacentric Rb(11.13)4Bnr chromosome. Its multivalent at meiosis I differs from the one in the former karyotype in one respect: The (acrocentric) chromosome 11 is here involved once separately and once as one of the arms of the metacentric, i.e. attached to the 13 arm, whereas in the former karyotype the two chromosomes 11 are involved each in a metacentric.

- For the short translocated segment 13 t , in both T(1;11.13S),+/+, Rb4Bnr and T70H,+/+,Rb4Bnr males, chiasma terminalization with proceeding meiosis I was observed less frequently compared with T70W+. It was argumented that this is probably due to spermatocyte I degeneration taking place to a higher extent in the former two karyotypes as in both, in comparison to T7OH/+, a greater frequency of meiosis 1 cells with short contracted bivalents was observed. The latter phenomenon was in an earlier study shown to correlate highly with a decreasing sperm count (4).

- In T(1;11.13S),+/+, Rb4Bnr a chiasmate association of the long interstitial segment 13 i was absent in 12.5% (N=1000) of the meiosis I cells, but only in 2.7% (N=1000) of these cells in T70H,+/+,Rb4Bnr males. A cause for this discrepancy could not be given the phenomenon could not be contributed to a precocious "disappearance" of the 13 i chiasma - and seems to be an effect of the metacentric chromosome present in homozygous condition in the former karyotype (4).

A significant increase of the 3:1 segregation of the multivalent chromosomes after anaphase 1 in comparison to T7OH/+ (3.5%) was found for T(1;11.13S),+/+,Rb4Bnr (10.9%) but most significantly for T70H,+/+,Rb4Bnr (22.6%). In the latter karyotype this refers only to the four chromosomes involved in that part of the multivalent caused by the T70H translocation. In T70H,+/+,Rb4Bnr, a preference of the small marker chromosome 1 13 to segregate ; with the metacentric Rb4Bnr chromosome was indicated and this could be sustained by the high frequency of Ts(1 13 )70H tertiary trisomics among the progeny of males of this karyotype. The results suggest that factors predisposing for 3:1 segregation and life expectancy of the tertiary trisomic product produced among the progeny may coincide (4).

In T70H,+/+,Rb4Bnr males the alternate and adjacent 1 segregation products could be distinguished at meiosis II and almost equal frequencies were found for both. In the offspring however, karyotypes resulting from adjacent 1 were found more frequently but this disagreement is probably due to the limited sample size of the offspring karyotyped (4).

In both T(1;11.13S),+/+,Rb4Bnr and T70H,+/+,Rb4Bnr male mice, the normal bivalent non-disjunction after anaphase I appeared to be highly elevated in comparison to +/+ and T70H/+ For the former karyotype a correlation was observed, moreover, between nondisjunction for the normal bivalents and adjacent 2 segregation of the multivalent chromosomes. This suggests either an interaction between the two phenomena or, alternatively, their coincidence may be an effect of the general (poor) physiological status of the cells. A sharp distinction between these alternatives cannot be made. The indication of spermatocyte I degeneration (see first point of this section) in both karyotypes puts weight on the latter hypothesis, assuming an interaction between the poorer physiological status of the cell and adjacent 2/normal bivalent non-disjunction.

A meiotic study of two translocations and a tertiary trisomic in the mouse (Mus musculus)
Boer, P. de - \ 1975
Landbouwhogeschool Wageningen. Promotor(en): J. Sybenga. - Wageningen : [s.n.] - 79
heterozygotie - genetische variatie - overerving - muizen - muridae - meiose - trisomie - heterozygosity - genetic variation - inheritance - mice - muridae - meiosis - trisomy
In this section, the order of the articles has not been closely followed. Each point ends with the number(s) of the article(s) (as given in the contents), where the conclusion is based on.

1) Cytological meiotic studies of T(2;8)26H and T(1;13)70H heterozygotes and Ts(1 13)70H tertiary trisomics indicate, that chiasmata are more often located in the distal (translocated) segments than in the proximal (interstitial) segments containing centric heterochromatin (3 and 5).

2) This study opens the possibility that the presence of centric heterochromatin decreases the probability of chiasma formation in its vicinity with a positive gradient distally (5).

3) The genetic lengths of the interstitial and translocated chromosome segments coincide rather well with the physical length of these segments as estimated with the aid of Giemsa-banding. This finding does not fit the tendency expressed in the conclusions 1 and 2. The apparent exception of this rule is segment 13 t which is overestimated when looking at genetic recombination. For cytological studies, the physical length of a segment is of a greater value (4).

4) Univalence for chromosome 1 13at metaphase I - anaphase I does not lead to an appreciable loss of this chromosome in the male, neither in the Ts(1 13)70H tertiary trisomic karyotype nor in the T(1;13)70H heterozygote (3 and 5).

5) In the T70H/+ karyotype, there is strong evidence for coorientation of the 1 13univalent so that the four reciprocal translocation involved chromosomes segregate two by two. Occasionally, equational separation of the two 1 13chromatids may occur at anaphase I (5).

6) The segregational behavior of heterozygous translocation multivalent configurations can, within the genetic background concerned, be best explained by time differences of chiasma terminalization during metaphase I - anaphase 1 (5).

7) The genetic background most likely exerts an influence on the behavior of mouse reciprocal translocations (5).

8) The reliability of the formula which relates the summed frequencies of adjacent II disjunction and numerical non- disjunction and the relative viability of heterozygous translocation outcross progeny depends on the existence of selection against small litters during gestation. This is the more likely when the theoretically expected litter size decreases (5).

9) A-chiasmate non-homologous chromosome association of the centric heterochromatin of chromosome 1 13and the X-chromosome does occur (3 and 5).

10) The majority of male Ts(1 13)70H tertiary trisomics are capable of producing offspring. Thus, tertiary trisomy does not invariably lead to sterility in the male mouse (2 and 3).

11) Tertiary trisomics for chromosome 1 13in the mouse display a variety of phenotypes. The condition can lead to death in utero, to death before weaning, to morphologically affected but viable animals and to animals with an unaltered appearance (2 and 3).

12) The ratio between morphologically affected and unaffected tertiary trisomics for chromosome 1 13at birth (live or dead) amounts to between 2 and 3. This ratio might depend on the genetic background concerned (2 and 3).

13) The most obvious abnormality of the morphologically affected tertiary trisomics of the Ts(1 13)70H karyotype is a malformation of the bones of the skull which often leads to an abnormal growth of the upper and lower incisors (2).

14) The impaired fertility of Ts(1 13)70H males is most probably due to a lowered production of functional spermatozoa and the consequences this has for the continuation of pregnancy. Thus, the elimination of "unbalanced" progeny is not the first cause (3).

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