Digestion 14: 269 280 (1976)
The Effect of a Single Dose of Cyclophosphamide on the Jejunum of Specified Pathogenfree and Germfree Rats1 R. Ecknauer and U. Lôhrs Department of Clinical Chemistry (Head: Prof. Dr. K. Rommel), University of Ulm. Ulm, and Institute of Pathology (Head: Prof. Dr. M. Eder), University of Munich. Munich
Key Words. Cyclophosphamide • Disaccharidascs ■Intestinal mucosa ■Jejunum ■RatsThymidine kinase • Villus Abstract. After a single dose of cyclophosphamide (100 mg/kg) cell proliferation in the jejunum of the rat decreased within the first 24 h and returned to the initial level after 48 h. tinder the influence of cyclophosphamide, an increased cell loss in germfree rats could be observed. Villus height and villus cell count tended to decrease. Changes in disaccharidasc activity in mucosal scrapings with respect to protein and DNA content could not be demon strated. An influence of presence or absence of bacterial fora could be observed.
The influence of cyclophosphamide (CY) on the small intestine is sum marized in table I. In general, the following alterations can be found: (1) inhi bition of cell regeneration; (2) direct damage to intestinal epithelial cells, and (3) decrease of the activity of brush-border enzymes. According to Rybak (41) and Achord (2), different responses to cytostatic treatment depending on the intestinal microflora can be expected. Results here presented should help answer the following two questions: (l)D o es CY in a single dose influence disaccharidase activity, cell turnover and morphology in the jejunum of the rat? (2) Is the influence dependent on the presence or absence of bacteria in the intestine?
' This study was supported by the ‘Deutsche l-orschungsgcmeinschaft’, Bonn (SI’B 112 E 4).
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Received: October I, 1975, accepted: January 9, 1976.
270
Ecknauer/Löltrs Table /. Effect of CY on morphology and cell turnover in the small intestine Species Site
Dosage mg/kg
Results
Authors
Man J
various
no significant light microscopic changes on villi
Wolff. 1967 (50)
Man J
100 s.d.
no significant ultrastructural changes (microvilli, mitochondria, nuclei)
Wolf et at. 1968 (49)
Mouse D, J, I
250 s.d. 30
3H-TdR index; decrease (to 41 % in 24 h) Ag grain count: decrease (to 33 % in 24 h) no effect
Palme et ai. 1963 (33)
Golden hamster J. I
400 800 s.d.
crypt: cytoplasm, inclusion bodies, nuclear alterations, loss of nuclei into crypt lumen villus: cytoplasm, inclusion bodies, nuclear alterations, decrease of AP (max. after 8 h)
Waldeck, 1972 (46)
Mouse D, J, I
2 X 50 14 days
crypt: like Waldeck (46) villus: villus top alterations, overaged cells changes of goblet cells and Paneth’s cells
Eisenhuth and Geyer, 1966 (11)
D = Duodenum; J = jejunum; I = ileum; s.d. = single dose; AP = alkaline phosphatase activity.
Methods Animals and Experimental Design Male germfree (GF) and specified pathogen-free (SPF) outbred VVistar rats (strain SV-49) from Thomae, Biberach were kept in Trexler plastic isolators for the duration of the experiment and got sterilized laboratory chow (Altromin 13/14 fortified. Altrogge, LageLippe) and sterile water ad libitum. After a period of adaptation of at least 1 week, the animals were weighed and injected subcutaneously with freshly prepared CY solution 100 mg/kg body weight or saline according to the following scheme: -4 8 h
-2 4 h
Day of investigation
Saline Saline CY
saline CY saline
control 24 h after CY 48 h after CY
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After the injection, the animals had no access to solid food, only water ad libitum. Coprophagy was reduced by wire-bottomed cages. The entire series of injections were given between 7 and 7.30 a.m. by the same individual. Anesthesia with sodium pentobarbital i.p.
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(65 mg/kg body weight) was initiated between 7.45 and 8.15 a.m. on the day of investiga tion. 6 animals (1 of each regimen) were investigated simultaneously (in each group there were 10 rats). A jejunal segment of about 20 cm was perfused for 3 h to determine cell loss. At the end of the perfusion period, the jejunal segment directly distal to the perfused segment was removed, rinsed with cold saline (154mmol/l NaCl) and the mucosa scraped off with a glass slide. One part of these scrapings was immediately frozen, freeze-dried, and then stored at 30°C. The other part was homogenized about 1 h after the animal was killed. This homogenate was the basis to measure the activity of thymidine kinase. Im mediately after sacrificing the rat, but prior to all manipulations, a 1-cm specimen of jejunum about 22 cm distal to the ligament of Treitz was removed for histology and fixed in Shaffer’s fluid (a buffered methanol-formalin-glucose solution). Reagents, Solutions and Instruments Reagents. Ethanol, Na2HP04, NaH2P04, MgCl2, Mg-titriplex, Tris, O. 1 N KOH, 1 N HCI, toluene (Merck, Darmstadt), ATP (Boehringer, Mannheim), thymidine (Serva, Heidel berg), ammonium formiate (Roth, Karlsruhe), bovine serum albumin (Behring, Marburg), 3H-thymidinc (specific activity 2 Ci/mmol) (Amersham Buchler, Braunschweig), Omnifluor (NEN Chemicals), Soluene-350 (Packard), DEAE-cellulose Whatman DE-81; all reagents were of analytic grade. Sterile Ringer solution (Pfrimmer, Erlangen) and water (Braun, Melsungen), trichloracetic acid (TC'A) (Merck, Darmstadt), pentobarbital natrium (Nembu tal; Abbott). The CY (Endoxan) was a generous gift from Asta-Werke AG. Solutions. For perfusion, a Ringer-glucose solution was prepared with a glucose con centration of 56 mmol/l using sterile solutions. The other solutions used were 50 % TCA, 5 % TCA, 15 % albumin solution, 0.001 N phosphate buffer pH 7.4, 1 mol Tris-HCI buffer pH 8.0. 220 mg ATP were dissolved in 8 ml 0.1 N KOH and adjusted to pH 7.0. For deter mining the activity of thymidine kinase, a thymidine solution was prepared with a final thymidine concentration of 1.4 umol/ml and a specific activity of 0.238 Ci/mmol. Instruments. Eppcndorf Microlitersystem and Eppendorf Substratmessplatz (Netheler 6 Hinz. Hamburg), perfusor. homogenizer type Potter S, calibrated homogenization vial and calibrated Teflon pestles (Braun, Melsungen). Perfusion The DNA loss was determined according to the following modification of the method of l.oehry et al. (27): only a jejunal segment of 20 cm was perfused with a glucose-con taining solution at a rate of 12 rnl/h. After perfusion, the length of the segment was mea sured by distending the gut with a 3-gram weight.
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Biochemical Methods Disaccharidase activity. The freeze-dried mucosal scrapings were homogenized 6 X 15 sec in the Potter homogenizer at 800 rpm cooled in an ice bath. Measurement of maltase (EC 3.2.1.20), sucrase (EC 3.2.1.26) and lactase (EC 3.2.1.23) activity was performed according to Dahlqvist (6) in an adaptation to the Eppendorf Microlitersystem (4). Thymidine kinase activity. The activity of thymidine kinase (EC 2.7.1.21) was deter mined in freshly homogenized tissue. The homogenate (procedure as for disaccharidase activity was centrifuged 30 min at 30,000 g at 4 °C. The supernatant was used to determine the enzyme activity according to Wiimanns (47). Protein and DNA. Protein content of the mucosal scrapings was determined according to l.owry, DNA content of the mucosal scrapings and the perfusate by the diphenylamine reaction according to Schneider in an adaptation to the Eppendorf Microlitersystem (8). These parameters were used as reference bases for enzyme activities.
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Bacteriology Lack of contamination was demonstrated by monitoring the isolator at least twice a week, using standard procedures (14, 43). The SPF rats are descendents of GF rats asso ciated 3 years ago with the following microbial flora: Lactobacillus acidophilus (ATCC 11506). Lactobacillus bifidus (isolated from human baby feces). Streptococcus lactis, Strep tococcus faecalis (ATCC 10541), Bacteroides symbiosum = Fusobacterium symbiosum (ATCC 14940) and Escherichia coli (isolated from rats). Histology, Morphometry The 4 -5 jum in parablast embedded sections were stained with Hi.. The quantitative evaluation of the sections was performed according to the following criteria: (1) on each slide, 11 crypt-villus systems were measured and counted. Only the cryptvillus systems were used which showed a continuity of crypt epithelium to villus epithelium. Only optimal vertically sectioned crypts and villi in their whole length were used; (2) all epithelial cells along the section of one crypt and the villus attached to it were counted. Also the number of intraepithelial leukocytes in the villus epithelium and the number of mitotic figures (metaphase up to telophase) in the crypt epithelium were counted. The mitotic index expresses the number of cells in mitosis as a percentage of the total crypt cells counted in the section and (3) with a micrometer scale, the height of villi and the depth of the crypts were measured separately and expressed in micrometers. Statistics The results were expressed as arithmetic means ± standard error. The statistical meth ods W'ere multifactorial variance analysis and variance analysis. In all tests, the p value of 0.05 has been applied as the limit of significance. If means are described as different, this level of significance was reached.
Results General Initial body weight (160 ± 30 g), absolute (27—33 g) as well as relative (16—20 % of initial body weight) weight loss were not different in the 6 groups (fig. 1). The cecal sac with contents was clearly heavier in the GF rats with no detectable influence of CY (fig. 1). In the SPF rats, there was not significantly (5 %p 10 %) higher incidence of macrohematuria (fig. 1).
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Cell Turnover The thymidine kinase activity 24 h after CY was at the control level but rose 48 h after CY (fig. 2 a) in the GF rats. The DNA content in the perfusate was, at least in the GF rats, elevated under CY (fig. 2b). The SPF rats seemed to behave differently, but technical difficulties, such as admixture of blood and mucus or obstructions limited almost exclusively to the SPF control group, do not allow a definite interpretation. In a pilot study, SPF rats showed a similar increase in DNA content of the perfusate under CY as the GF rats do in the present study.
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Fig. 1. Changes in body weight and cecal weight under the influence of a single dose (100 mg/kg body weight) CY. On the first line, the number of animals with macroscopically detectable hematuria, below this the number of animals in each group. O = Controls; 24 = the animals 24 h after CY, 48 = the animals 48 h after CY.
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Morphology Control. Measured crypt depth and the crypt cell count were not different in GF and SPF rats. Villus height and villus cell count was higher in the GF rats. There was no difference between GF and SPF rats in the number of mitotic figures per crypt section or in the mitotic index (table II). 24 h after CY. Both the GF and SPF rats showed fewer cells per crypt section but no difference in crypt depth, a decrease in mitotic index and number of mitotic figures per crypt. In the GF rats, the villi seemed to become smaller having fewer cells per villus section. This tendency could not be detected in the SPF rats (table II). 48 h after CY. In both GF and SPF rats, crypt cell count, number of mitotic figures and the mitotic index increased the values reaching the control level, and in some instances, seemed to exceede this level. The villi in both groups appeared to be smaller and to have fewer cells than in the control rats (table II). Intraepithelial leukocytes. The number of intraepithelial leukocytes per vil lus section, as well as the relative number of intraepithelial leukocytes (percent of the total cell count per villus section) is lower in the GF rats than in the SPF rats. There seemed to be a tendency to decrease under the influence of cyclo phosphamide (table II).
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Fig. 2. a Activity of thymidine kinase after 100 mg/kg CY. The activity is expressed in nanomoles thymidine phosphorylated per gram mucosal protein per minute, b Cell loss 24 and 48 h after CY (100 mg/kg body weight). Cell loss is expressed in micrograms DNA in the intestinal perfusate per hour per centimeter gut length distended by a 3-grant weight. The last three columns are the results of a pilot study. They are not directly comparable to the data of the present study, because those rats were not fasted and the cell loss is related to the gut length not distended by a 3-gram weight.
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Disaccharidase Activity Maltase, sucrase and lactase activity with reference to protein as well as to DNA content of mucosal scrapings was not changed significantly by CY. The ‘specific activities’ of maltase and lactase but not that of sucrase were higher in the mucosal scrapings of the GF rats (fig. 3).
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“ 111 Hi O
GF
S PF
GF
MALTASE
SPF
24
48
O
GF
24
48
SPF LACTASE
SUCRASE
n o m g / k g t.c cyctophosphamM
Fig. 3. Influence of CY on the disaccharidase activity in the mucosa of rat jejunum. Each group consists of 10 rats.
Table II. Quantitative evaluation of the morphology of crypts and villi Morphology
GF rats controls
Villi Height, pm 507 ± 59 Number of cnterocytes 192 ± 25 Number of intraepithelial leukocytes 15 ± 3 Crypts Depth, pm 124 ± 16 Number of cnterocytes 49 ± 5 Number of intraepithelial leukocytes 2.1 ± 0.5
SPF rats 24 h after CY
48 h after CY
controls
24 h after CY
48 h after CY
458 ± 18 182 ± 5
447 ± 27 177 ± 10
382 ± 61 169 t 27
424 i 45 168 ± 20
389 i 40 149 * 12
14 ± 1
12 ± 2
21 ± 7
19 i 4
17 ± 2
112 t 8 41 ± 4
115 ± 11 46 ± 3
129 ± 17 52 i 7
120 ± 20 45 ± 4
125 ± 15 49 t 5
1.4 ± 0.8
2.3
2.1 ± 0.4
1.7 ± 0.4
2.5 ± 0.6
î
0.5
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Results are arithmetic means t standard deviation from 6 animals of each group.
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Discussion
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The incidence of macrohematuria is a sign of a hemorrhagic cystitis (26,35, 36, 40). More detailed studies are necessary to clarify whether there is a dif ference in damage to tire bladder epithelium depending on the intestinal micro flora. The attempt at describing the cell loss by measuring the DNA content in the perfusion fluid in the present modification was not entirely satisfactory, but in spite of these limitations, there seemed to be an increased DNA loss under the influence of CY in GF (present investigation) and SPF rats (previous un published results). This increased DNA loss may represent an increased cell loss, most probably at the villus tops but perhaps from the crypts too (9, 11, 18,31, 37). From this investigation it cannot be stated whether or not there is a dif ference in cell loss in SPF and GF rats. The activity of thymidine kinase was used as a criterion of DNA synthesis and therefore as a measure of cell proliferation as in hematological investigations (48, 49). Thymidine kinase activity is detectable solely in crypt cells, never in villus cells (22, 23) and is not of bacterial origin (23). Autoradiographic investi gations have showed that CY decreased the regenerative pool in the crypt as well as DNA synthesis in the remaining cells (33, 34). Both effects will reduce the thymidine kinase activity. Helge et al. (21) and Oberdisse (31) observed an increase in thymidine kinase activity in tumors under the influence of a-hexachlorohexan which could be suppressed by CY. The attempt at gaining insight into the cell renewal by measuring the activity of thymidine kinase seems to be indicated by these findings. The value of thymidine kinase activity as a valid indication of cell proliferation in the epithelium of the small intestine has to be established by a comparison with the results obtained with conventional meth ods of measuring cell production. Under the assumption that thymidine kinase activity represents the cell production in some way, the present data indicate an increase of cell production 48 h after CY. This is advocated by the increase in mitotic figures per crypt section and mitotic index at the same time. A decrease in thymidine kinase activity could not be observed. The decrease in crypt depth, crypt cell count and mitotic frequency reflects the damage to the regenerative cell population and is described after a single high dose of various cytostatics (3, 9, 30, 33, 39). The decrease in villus height and villus cell count is also consistant with the results after a single dose of various cytostatics (3, 9, 30, 38, 39). The tendency of the villi in GF rats to become smaller at an earlier stage than in the SPF rats cannot be explained satisfactorily. Possibly, the higher dose the GF rats received (CY was related to the body weight not corrected for weight of cecum with contents) may have some bearing on this. From the results of Gustafsson and Persson (17) and Einarsson et al. (10) it does not seem warranted to assume a reduced ability to metabolize CY in
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GF rat livers. In conformity with Galjaard et al. (15) and Levenson (25), no difference in crypt cell count and mitotic frequency between SPF and GF rats could be detected. This is in contradiction with results that indicate a higher proliferation in the crypts of ‘conventional’ animals (1, 16, 29, 44). These dif ferences may be due to the 48-hour starvation in this investigation, strain dif ferences (20), the composition of the microbial flora in the SPF rats (29) and to the fact that the GF rats were not compared with ‘conventional’ rats but with SPF rats (5). The number of intraepithelial leukocytes is not influenced by the treatment. In agreement with Otto and Lewerenz (32) but in contrast with Fichtelius (12), there were fewer leukocytes situated between the epithelial cells in the jejunum of the GF rat. Although most observers have described a decrease in disaccharidase activity under the influence of cytostatics (19), the results presented here do not support this conclusion. The following reasons should be discussed regarding this discrep ancy: (1) Dose: the dose of 100mg/kg body weight is not a high dose, but suf ficient to influence the hemopoietic system (35, 45), the bladder epithelium (35) and the turnover of the intestinal epithelium (34). Perphaps disaccharidase activity is influenced only by higher doses or has recovered already at these time intervals. (2) Application: it seems unlikely that the subcutaneous application in the present investigation had an influence on the results (42). (3) Reference system: in the experiments of ae Both et al. (7) and Menge et al. (28), significant changes in villus height were not accompanied by corre sponding alterations in disaccharidase activity referred to protein. In tírese situa tions and perhaps in the present experiments, changes in enzyme activity were accompanied by similar alterations in the reference system (mucosal protein) and as a consequence of this, the specific activity of the enzyme remained unaltered. (4) Fasting: the period of fasting (48 h) is apt to change all parameters investigated in this study — for instance disaccharidase activity, protein and DNA content of the mucosa. (5) Control animals: in the present experiments, the control animals were subjected to tire same procedures as the test animals. Therefore influences of the manipulations and experimental conditions can be excluded.
The authors wish to thank W. Calvo (Abteilung für Klinische Physiologie. Universität Ulm) for preparing the histological specimens. W. Gaus (Abteilung für Medizinische Stati stik, Universität Ulm) for performing the statistical analysis, 11. Meyer (Sektion Gnoto-
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A cknowledgemen ts
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biotik, Universität Ulm) for his advice in GF technique and for confirming the GF status of the isolators, and K. Wilms (Medizinische Universitätsklinik, Tübingen) for his help in intro ducing the measurement of thymidine kinase activity. We also thank Ms. I. Schnösenberg for competent technical assistance.
References
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1 Abrams, D.G.; Bauer, H., and Sprinz, H.: Influence of the normal flora on mucosal morphology and cellular renewal in the ileum. A comparison of germfree and conven tional mice. Lab. Invest. 12: 355 364 (1963). 2 Achord, J.L.: The effect of methotrexate on rabbit intestinal mucosal enzymes and flora. Am. J. dig. Dis. 14: 315-323 (1969). 3 Altmann, G.G.: Changes in the mucosa of the smaL intestine following methotrexate administration or abdominal X-irradiation. Am. J. Anat. 140: 263-280 (1974). 4 Böhmer, R.: Rommel, K.; Goberna, R. und Raptis, S.: Disaceharidasenaktivität des Jejunums subtotal pankreatektomierter Ratten. Clinica chim. A ctaJJ: 61 -67 (1971). 5 Clarke, R.M.: Diet, mucosal architecture and epithelial cell production in the small intestine of specified-pathogen-free and conventional rats. Lab. Anim. Care 9: 201-209 (1975). 6 Dahlqvist, A.: Assay of intestinal disaccharidases. Enzymol. biol. clin. 11: 52 66 (1970). 7 De Both, N.J.; Van Dongen, J.M.; Van Hofwegen, B.; Keulemans, J.; Visser, W.J., and Galjaard, H.: The influence of various cell kinetic conditions on functional differentia tion in the small intestine of the rat. A study of enzymes bound to subcellular organ elles. J. develop. Biol. 38: 119-137 (1974). 8 Ecknauer, R. und Schnösenberg, 1.: Teilmechanisierte Bestimmung des Proteins und der Desoxyribonucleinsäure im Mikrolitermassstab in der Dünndarmschleimhaut. Z. Gastroent. (in press, 1975). 9 Eder, M.; Rostock, H. und Vogel, G.: Die Wirkung von Folsäureantagonisten (Methotrexat) auf die Regeneration der Darmschleimhaut. Virchows Arch. path. Anat. Physiol. 341: 164-176(1966). 10 Einarsson, K.; Gustafsson, J.A., and Gustafsson, B.E.: Liver microsomal hydroxylation of steroid hormones after establishing an indigenous microflora in germfree rats. Proc. Soc. exp. Biol. Med. 145: 48-52 (1974). 11 Eisenhuth, I. und Geyer, G.: Veränderungen am Dünndarmepithe! der Albinomaus durch chronische Behandlung mit den N-Iost Stoffen Endoxan, Trimitan und Degranol unter besonderer Berücksichtigung der Paneth’schen Körnerzcllcn. Acta histochem. 25: 71-85 (1966). 12 Fichtelius, K.E.: The gut epithelium a first level lymphoid organ? Expl Cell Res. 49: 87-104 (1968). 13 Fortin-Magana, R.; Hurwitz, R.; Herbst, J.J., and Kretchmer, N.: Intestinal enzymes: indicators of proliferation and differentiation in the jejunum. Science, N.Y. 167: 1627-1628 (1970). 14 Fuller, R.: The routine microbiological control of germfree isolators; in Coates The germfree animal in research (Academic Press, London 1968). 15 Galjaard. H.; Meer-Fieggen, W. van der, and Giesen, J.: Feedback control by functional villus cells on cell proliferation and maturation in intestinal epithelium. Expl Cell Res. 73: 197-207 (1972). 16 Guenet, J.L.: Sacquet, E.: Gueneau, G. et Meslin, J C.: Action de la microflora totale
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du rat sur l’activité mitotique des cryptes de Lieberkühn. C.r. hebd. Séanc. Acad. Sei., Paris 270: 3087 3090 (1970). Gustafsson, B.E. and Persson, A.: Reduced sleeping time in germfrec rats after pento barbital administration. 5th Int. Symp. on Gnotobiology, Stockholm 1975. Hampton, J.C.: A comparison of the effects of X-irradiation and colchicine on the intestinal mucosa of the mouse. Radiat. Res. 28: 37- 59 (1966). Hartwich, G.: Disaccharidasen der Dünndarmschleimhaut unter zytostatischer Behand lung; Habilitationsschrift Erlangen (1974). Heit, H.: Intestinal renewal system in gnotobiotic mice and rats (unpublished, 1973). Helge, H.; Oherdisse, E. und Engels, K.: Hemmbarkeit der DNA-polymerase in Zell kernen und Mitochondrion von Leber- und Tumorgewebe durch Cyclophosphamid. Arch. Pharmakol. exp. Path. 260: 139-140 (1968). Herbst, J.J.; Fortin-Magana, R., and Sunshine, P.: Relationship of pyrimidine biosyn thetic enzymes to cellular proliferation in rat intestine during development. Gastro enterology 59: 240 246 (1970). lemhoff, W.G.J.: Van den Berg, J.W.O.: De Pijper, A.M., and Hülsmann, W.C.: Meta bolic aspects of isolated cells from rat small intestinal epithelium. Biochim. biophys. Acta 2/5: 229-241 (1970). Imondi, A.R.: Balis, M.E., and Lipkin, M.: Changes in enzyme levels accompanying differentiation of intestinal epithelial cells. Expl Cell Res. 58: 323 330 ( 1969). Levenson, S.: Personal commun. Locher, G. W. and Cooper, E.H.: Repair of rat urinary bladder epithelium following injury by cyclophosphamide. Investve Urol. 8: 116-1 23 (1970). Loehry, C.A.: Croft, D.N.: Singh, A.K., and Creamer, B.: Cell turnover in the rat small intestinal mucosa: an appraisal of cell loss. Gut 10: 13-18 (1969). Menge, H.; Gräfe, M.; Lorenz-Meyer, H., and Riechen, E.O.: The influence of food intake on the development of structural and functional adaptation following ileal resection in the rat. Gut 16: 468-472 (1975). Meslin, J.-C.; Sacquet, E. et Raibaud, P.: Action d’une flora microbienne qui ne déconjugue pas les sels biliaires sur la morphologie et le renouvellement cellulaire de la muqueuse de l’intestin grêle du rat. Annls Biol. anim. Biochim. Biophys. 14: 709-720 (1974). Millington, P.F.; Finean, J.B.: Forbes, O.C., and Frazer, A.C.: Studies of the effects of aminopterin on the small intestine of rats. 1. The morphological changes following a single dose of aminopterin. Expl Cell Res. 28: 162-178 (1962). Oherdisse, E.: Pharmakologische Beeinflussbarkeit der Induktion von Fermenten im Pyrimidinstoffwechsel. Arch. Pharmakol. exp. Path. 260: 182 183 (1968). Otto, H.F. und Lewerenz, 1.: Untersuchungen zur Ultrastruktur des Dünndarms keim frei aufgezogener FW 49 Ratten. 1. Epitheliale Befunde unter besonderer Berücksichti gung der Paneth-Zellen. Virchows Arch. Abt. A. Path. Anal. 360: 235 251 (1973). Palme, G.; Liss, E.: O eff K. und Platis, A.: Der Einfluss von Cyclophosphamid auf die DNS-Synthese von normalen proliferierenden Zellen sowie Ascites-Tumorzellen. Arzneimittel-Forsch. 13: 1034-1039(1963). Palme, G.; Liss, E. und Wiebel, F : Autoradiographische Untersuchungen über das Verhalten einzelner Interphasenabschnitte des Dünndarmepithels der Maus unter der Einwirkung von Cyclophosphamid. Naturwissenschaften 51: 197 198 (1964). Philips, F.S.; Sternberg, S.S.; Cronin, A.P., and Vidal, A.P.: Cyclophosphamide and urinary bladder toxicity. Cancer Res. 21: 1577-1589 (1961). Primack, A.: Amelioration of cyclophosphamide-induced cystitis. J. natn. Cancer Inst. 47: 223-227 (1971). Downloaded by: Univ. of California Santa Barbara 128.111.121.42 - 3/7/2018 4:31:03 AM
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Raymackers, E. and Hugon, J.S.: Morphological and cytochomical modifications induced by colchicine treatment in the duodenal epithelium of mouse. Histochemie 34: 65-76 (1973). 38 Redgrave, T.G. and Simmonds, W.J.: Effect of aminopterin on the absorption of fat into the lymph of unanesthetized rats. Gastroenterology 52: 54-66 (1967). 39 Roche, A.C.; Bognel, J.Cl.: Bognel, C, and Bernier, J.J.: Correlation between histologi cal changes and glucose intestinal absorption following a single dose of 5-fluorouracil. Digestion 3: 195-212 (1970). 40 Rubin, J.S. and Rubin. R.T.: Cyclophosphamide hemorrhagic cystitis. J. Urol. 96: 313-316 (1966). 41 Rybak, B.J.: Electron microscopic studies of intestinal lesions. I. Aminopterin-induced lesions in mice. Gastroenterology 42: 306-318 (1962). 42 Schmidt, D. und Hayn, J.: Verschiedene Applikationsmöglichkeiten von Zytostatika zur intra- und postoperativen Metastasen- und Rezidivprophylaxe in i'orm einer Stosstherapie. Arch. klin. Chir. 324: 32-43 (1969). 43 Subcommittee on Standards for Gnotobiotes: Gnotobiotes (National Academy of Science, Washington 1970). 44 Sugiyama. T. and Stewart, W.F.: Mitotic indices of the intestinal epithelium in germfree rats and in conventional rats enterally exposed to germfree rat cecal contents. 5th Int. Symp. on Gnotobiology. Stockholm 1975. 45 Tyler, W.S.: Niskanen, E.; Stohlman, F.: Keane, J., and Howard, D.: The effect of neutropenia on myeloid growth and the stem cell in an in vivo culture system. Blood 40: 634-645 (1972). 46 Watdeck, H.H.: Die Diinndarmschleimhaut nach hohen Dosen Cyclophosphamid. Mor phologische und histochemischc Untersuchungen beim Goldhamster. Z. Gastroent. 10: 543-554 (1972). 47 Wilmanns, W.: Die Thymidinkinase in normalen und leukämischen menschlichen Blut zöllen. Klin. Wschr. 45: 505-511 (1967). 48 Wilms, K. und Wilmanns, W.: Die Beurteilung der Therapie akuter Leukosen mit zyto toxischen Substanzen durch Untersuchungen von Enzymen der DNS-Synthese in den Leukozyten und in der Zellkultur; in Wüst Aktuelle Probleme der Therapie maligner Tumoren, pp. 96-108 (Thieme, Stuttgart 1973). 49 Wolf, M Ley, I. und Klein, K.: Der Einfluss von Cyclophosphamid auf die Ultrastruk tur der Diinndarmschleimhaut. Zentbl. inn. Med. 23: 727 (1968). 50 Wolff G.: Morphologische Veränderungen der Dünndarmmukosa unter Zytostatika behandlung. Ber. Ges. inn. Med. 5: 159-160 (1967). 37
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Dr. R. Ecknauer, Departement für Klinische Chemie, Steinhövelstr. 9, O 79 Ulm (ERG)
The Effect of a Single Dose of Cyclophosphamide on the Jejunum of Specified Pathogenfree and Germfree Rats1 R. Ecknauer and U. Lôhrs Department of Clinical Chemistry (Head: Prof. Dr. K. Rommel), University of Ulm. Ulm, and Institute of Pathology (Head: Prof. Dr. M. Eder), University of Munich. Munich
Key Words. Cyclophosphamide • Disaccharidascs ■Intestinal mucosa ■Jejunum ■RatsThymidine kinase • Villus Abstract. After a single dose of cyclophosphamide (100 mg/kg) cell proliferation in the jejunum of the rat decreased within the first 24 h and returned to the initial level after 48 h. tinder the influence of cyclophosphamide, an increased cell loss in germfree rats could be observed. Villus height and villus cell count tended to decrease. Changes in disaccharidasc activity in mucosal scrapings with respect to protein and DNA content could not be demon strated. An influence of presence or absence of bacterial fora could be observed.
The influence of cyclophosphamide (CY) on the small intestine is sum marized in table I. In general, the following alterations can be found: (1) inhi bition of cell regeneration; (2) direct damage to intestinal epithelial cells, and (3) decrease of the activity of brush-border enzymes. According to Rybak (41) and Achord (2), different responses to cytostatic treatment depending on the intestinal microflora can be expected. Results here presented should help answer the following two questions: (l)D o es CY in a single dose influence disaccharidase activity, cell turnover and morphology in the jejunum of the rat? (2) Is the influence dependent on the presence or absence of bacteria in the intestine?
' This study was supported by the ‘Deutsche l-orschungsgcmeinschaft’, Bonn (SI’B 112 E 4).
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Received: October I, 1975, accepted: January 9, 1976.
270
Ecknauer/Löltrs Table /. Effect of CY on morphology and cell turnover in the small intestine Species Site
Dosage mg/kg
Results
Authors
Man J
various
no significant light microscopic changes on villi
Wolff. 1967 (50)
Man J
100 s.d.
no significant ultrastructural changes (microvilli, mitochondria, nuclei)
Wolf et at. 1968 (49)
Mouse D, J, I
250 s.d. 30
3H-TdR index; decrease (to 41 % in 24 h) Ag grain count: decrease (to 33 % in 24 h) no effect
Palme et ai. 1963 (33)
Golden hamster J. I
400 800 s.d.
crypt: cytoplasm, inclusion bodies, nuclear alterations, loss of nuclei into crypt lumen villus: cytoplasm, inclusion bodies, nuclear alterations, decrease of AP (max. after 8 h)
Waldeck, 1972 (46)
Mouse D, J, I
2 X 50 14 days
crypt: like Waldeck (46) villus: villus top alterations, overaged cells changes of goblet cells and Paneth’s cells
Eisenhuth and Geyer, 1966 (11)
D = Duodenum; J = jejunum; I = ileum; s.d. = single dose; AP = alkaline phosphatase activity.
Methods Animals and Experimental Design Male germfree (GF) and specified pathogen-free (SPF) outbred VVistar rats (strain SV-49) from Thomae, Biberach were kept in Trexler plastic isolators for the duration of the experiment and got sterilized laboratory chow (Altromin 13/14 fortified. Altrogge, LageLippe) and sterile water ad libitum. After a period of adaptation of at least 1 week, the animals were weighed and injected subcutaneously with freshly prepared CY solution 100 mg/kg body weight or saline according to the following scheme: -4 8 h
-2 4 h
Day of investigation
Saline Saline CY
saline CY saline
control 24 h after CY 48 h after CY
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After the injection, the animals had no access to solid food, only water ad libitum. Coprophagy was reduced by wire-bottomed cages. The entire series of injections were given between 7 and 7.30 a.m. by the same individual. Anesthesia with sodium pentobarbital i.p.
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(65 mg/kg body weight) was initiated between 7.45 and 8.15 a.m. on the day of investiga tion. 6 animals (1 of each regimen) were investigated simultaneously (in each group there were 10 rats). A jejunal segment of about 20 cm was perfused for 3 h to determine cell loss. At the end of the perfusion period, the jejunal segment directly distal to the perfused segment was removed, rinsed with cold saline (154mmol/l NaCl) and the mucosa scraped off with a glass slide. One part of these scrapings was immediately frozen, freeze-dried, and then stored at 30°C. The other part was homogenized about 1 h after the animal was killed. This homogenate was the basis to measure the activity of thymidine kinase. Im mediately after sacrificing the rat, but prior to all manipulations, a 1-cm specimen of jejunum about 22 cm distal to the ligament of Treitz was removed for histology and fixed in Shaffer’s fluid (a buffered methanol-formalin-glucose solution). Reagents, Solutions and Instruments Reagents. Ethanol, Na2HP04, NaH2P04, MgCl2, Mg-titriplex, Tris, O. 1 N KOH, 1 N HCI, toluene (Merck, Darmstadt), ATP (Boehringer, Mannheim), thymidine (Serva, Heidel berg), ammonium formiate (Roth, Karlsruhe), bovine serum albumin (Behring, Marburg), 3H-thymidinc (specific activity 2 Ci/mmol) (Amersham Buchler, Braunschweig), Omnifluor (NEN Chemicals), Soluene-350 (Packard), DEAE-cellulose Whatman DE-81; all reagents were of analytic grade. Sterile Ringer solution (Pfrimmer, Erlangen) and water (Braun, Melsungen), trichloracetic acid (TC'A) (Merck, Darmstadt), pentobarbital natrium (Nembu tal; Abbott). The CY (Endoxan) was a generous gift from Asta-Werke AG. Solutions. For perfusion, a Ringer-glucose solution was prepared with a glucose con centration of 56 mmol/l using sterile solutions. The other solutions used were 50 % TCA, 5 % TCA, 15 % albumin solution, 0.001 N phosphate buffer pH 7.4, 1 mol Tris-HCI buffer pH 8.0. 220 mg ATP were dissolved in 8 ml 0.1 N KOH and adjusted to pH 7.0. For deter mining the activity of thymidine kinase, a thymidine solution was prepared with a final thymidine concentration of 1.4 umol/ml and a specific activity of 0.238 Ci/mmol. Instruments. Eppcndorf Microlitersystem and Eppendorf Substratmessplatz (Netheler 6 Hinz. Hamburg), perfusor. homogenizer type Potter S, calibrated homogenization vial and calibrated Teflon pestles (Braun, Melsungen). Perfusion The DNA loss was determined according to the following modification of the method of l.oehry et al. (27): only a jejunal segment of 20 cm was perfused with a glucose-con taining solution at a rate of 12 rnl/h. After perfusion, the length of the segment was mea sured by distending the gut with a 3-gram weight.
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Biochemical Methods Disaccharidase activity. The freeze-dried mucosal scrapings were homogenized 6 X 15 sec in the Potter homogenizer at 800 rpm cooled in an ice bath. Measurement of maltase (EC 3.2.1.20), sucrase (EC 3.2.1.26) and lactase (EC 3.2.1.23) activity was performed according to Dahlqvist (6) in an adaptation to the Eppendorf Microlitersystem (4). Thymidine kinase activity. The activity of thymidine kinase (EC 2.7.1.21) was deter mined in freshly homogenized tissue. The homogenate (procedure as for disaccharidase activity was centrifuged 30 min at 30,000 g at 4 °C. The supernatant was used to determine the enzyme activity according to Wiimanns (47). Protein and DNA. Protein content of the mucosal scrapings was determined according to l.owry, DNA content of the mucosal scrapings and the perfusate by the diphenylamine reaction according to Schneider in an adaptation to the Eppendorf Microlitersystem (8). These parameters were used as reference bases for enzyme activities.
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Bacteriology Lack of contamination was demonstrated by monitoring the isolator at least twice a week, using standard procedures (14, 43). The SPF rats are descendents of GF rats asso ciated 3 years ago with the following microbial flora: Lactobacillus acidophilus (ATCC 11506). Lactobacillus bifidus (isolated from human baby feces). Streptococcus lactis, Strep tococcus faecalis (ATCC 10541), Bacteroides symbiosum = Fusobacterium symbiosum (ATCC 14940) and Escherichia coli (isolated from rats). Histology, Morphometry The 4 -5 jum in parablast embedded sections were stained with Hi.. The quantitative evaluation of the sections was performed according to the following criteria: (1) on each slide, 11 crypt-villus systems were measured and counted. Only the cryptvillus systems were used which showed a continuity of crypt epithelium to villus epithelium. Only optimal vertically sectioned crypts and villi in their whole length were used; (2) all epithelial cells along the section of one crypt and the villus attached to it were counted. Also the number of intraepithelial leukocytes in the villus epithelium and the number of mitotic figures (metaphase up to telophase) in the crypt epithelium were counted. The mitotic index expresses the number of cells in mitosis as a percentage of the total crypt cells counted in the section and (3) with a micrometer scale, the height of villi and the depth of the crypts were measured separately and expressed in micrometers. Statistics The results were expressed as arithmetic means ± standard error. The statistical meth ods W'ere multifactorial variance analysis and variance analysis. In all tests, the p value of 0.05 has been applied as the limit of significance. If means are described as different, this level of significance was reached.
Results General Initial body weight (160 ± 30 g), absolute (27—33 g) as well as relative (16—20 % of initial body weight) weight loss were not different in the 6 groups (fig. 1). The cecal sac with contents was clearly heavier in the GF rats with no detectable influence of CY (fig. 1). In the SPF rats, there was not significantly (5 %p 10 %) higher incidence of macrohematuria (fig. 1).
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Cell Turnover The thymidine kinase activity 24 h after CY was at the control level but rose 48 h after CY (fig. 2 a) in the GF rats. The DNA content in the perfusate was, at least in the GF rats, elevated under CY (fig. 2b). The SPF rats seemed to behave differently, but technical difficulties, such as admixture of blood and mucus or obstructions limited almost exclusively to the SPF control group, do not allow a definite interpretation. In a pilot study, SPF rats showed a similar increase in DNA content of the perfusate under CY as the GF rats do in the present study.
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Fig. 1. Changes in body weight and cecal weight under the influence of a single dose (100 mg/kg body weight) CY. On the first line, the number of animals with macroscopically detectable hematuria, below this the number of animals in each group. O = Controls; 24 = the animals 24 h after CY, 48 = the animals 48 h after CY.
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Morphology Control. Measured crypt depth and the crypt cell count were not different in GF and SPF rats. Villus height and villus cell count was higher in the GF rats. There was no difference between GF and SPF rats in the number of mitotic figures per crypt section or in the mitotic index (table II). 24 h after CY. Both the GF and SPF rats showed fewer cells per crypt section but no difference in crypt depth, a decrease in mitotic index and number of mitotic figures per crypt. In the GF rats, the villi seemed to become smaller having fewer cells per villus section. This tendency could not be detected in the SPF rats (table II). 48 h after CY. In both GF and SPF rats, crypt cell count, number of mitotic figures and the mitotic index increased the values reaching the control level, and in some instances, seemed to exceede this level. The villi in both groups appeared to be smaller and to have fewer cells than in the control rats (table II). Intraepithelial leukocytes. The number of intraepithelial leukocytes per vil lus section, as well as the relative number of intraepithelial leukocytes (percent of the total cell count per villus section) is lower in the GF rats than in the SPF rats. There seemed to be a tendency to decrease under the influence of cyclo phosphamide (table II).
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274
Fig. 2. a Activity of thymidine kinase after 100 mg/kg CY. The activity is expressed in nanomoles thymidine phosphorylated per gram mucosal protein per minute, b Cell loss 24 and 48 h after CY (100 mg/kg body weight). Cell loss is expressed in micrograms DNA in the intestinal perfusate per hour per centimeter gut length distended by a 3-grant weight. The last three columns are the results of a pilot study. They are not directly comparable to the data of the present study, because those rats were not fasted and the cell loss is related to the gut length not distended by a 3-gram weight.
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Disaccharidase Activity Maltase, sucrase and lactase activity with reference to protein as well as to DNA content of mucosal scrapings was not changed significantly by CY. The ‘specific activities’ of maltase and lactase but not that of sucrase were higher in the mucosal scrapings of the GF rats (fig. 3).
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“ 111 Hi O
GF
S PF
GF
MALTASE
SPF
24
48
O
GF
24
48
SPF LACTASE
SUCRASE
n o m g / k g t.c cyctophosphamM
Fig. 3. Influence of CY on the disaccharidase activity in the mucosa of rat jejunum. Each group consists of 10 rats.
Table II. Quantitative evaluation of the morphology of crypts and villi Morphology
GF rats controls
Villi Height, pm 507 ± 59 Number of cnterocytes 192 ± 25 Number of intraepithelial leukocytes 15 ± 3 Crypts Depth, pm 124 ± 16 Number of cnterocytes 49 ± 5 Number of intraepithelial leukocytes 2.1 ± 0.5
SPF rats 24 h after CY
48 h after CY
controls
24 h after CY
48 h after CY
458 ± 18 182 ± 5
447 ± 27 177 ± 10
382 ± 61 169 t 27
424 i 45 168 ± 20
389 i 40 149 * 12
14 ± 1
12 ± 2
21 ± 7
19 i 4
17 ± 2
112 t 8 41 ± 4
115 ± 11 46 ± 3
129 ± 17 52 i 7
120 ± 20 45 ± 4
125 ± 15 49 t 5
1.4 ± 0.8
2.3
2.1 ± 0.4
1.7 ± 0.4
2.5 ± 0.6
î
0.5
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Results are arithmetic means t standard deviation from 6 animals of each group.
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Discussion
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The incidence of macrohematuria is a sign of a hemorrhagic cystitis (26,35, 36, 40). More detailed studies are necessary to clarify whether there is a dif ference in damage to tire bladder epithelium depending on the intestinal micro flora. The attempt at describing the cell loss by measuring the DNA content in the perfusion fluid in the present modification was not entirely satisfactory, but in spite of these limitations, there seemed to be an increased DNA loss under the influence of CY in GF (present investigation) and SPF rats (previous un published results). This increased DNA loss may represent an increased cell loss, most probably at the villus tops but perhaps from the crypts too (9, 11, 18,31, 37). From this investigation it cannot be stated whether or not there is a dif ference in cell loss in SPF and GF rats. The activity of thymidine kinase was used as a criterion of DNA synthesis and therefore as a measure of cell proliferation as in hematological investigations (48, 49). Thymidine kinase activity is detectable solely in crypt cells, never in villus cells (22, 23) and is not of bacterial origin (23). Autoradiographic investi gations have showed that CY decreased the regenerative pool in the crypt as well as DNA synthesis in the remaining cells (33, 34). Both effects will reduce the thymidine kinase activity. Helge et al. (21) and Oberdisse (31) observed an increase in thymidine kinase activity in tumors under the influence of a-hexachlorohexan which could be suppressed by CY. The attempt at gaining insight into the cell renewal by measuring the activity of thymidine kinase seems to be indicated by these findings. The value of thymidine kinase activity as a valid indication of cell proliferation in the epithelium of the small intestine has to be established by a comparison with the results obtained with conventional meth ods of measuring cell production. Under the assumption that thymidine kinase activity represents the cell production in some way, the present data indicate an increase of cell production 48 h after CY. This is advocated by the increase in mitotic figures per crypt section and mitotic index at the same time. A decrease in thymidine kinase activity could not be observed. The decrease in crypt depth, crypt cell count and mitotic frequency reflects the damage to the regenerative cell population and is described after a single high dose of various cytostatics (3, 9, 30, 33, 39). The decrease in villus height and villus cell count is also consistant with the results after a single dose of various cytostatics (3, 9, 30, 38, 39). The tendency of the villi in GF rats to become smaller at an earlier stage than in the SPF rats cannot be explained satisfactorily. Possibly, the higher dose the GF rats received (CY was related to the body weight not corrected for weight of cecum with contents) may have some bearing on this. From the results of Gustafsson and Persson (17) and Einarsson et al. (10) it does not seem warranted to assume a reduced ability to metabolize CY in
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GF rat livers. In conformity with Galjaard et al. (15) and Levenson (25), no difference in crypt cell count and mitotic frequency between SPF and GF rats could be detected. This is in contradiction with results that indicate a higher proliferation in the crypts of ‘conventional’ animals (1, 16, 29, 44). These dif ferences may be due to the 48-hour starvation in this investigation, strain dif ferences (20), the composition of the microbial flora in the SPF rats (29) and to the fact that the GF rats were not compared with ‘conventional’ rats but with SPF rats (5). The number of intraepithelial leukocytes is not influenced by the treatment. In agreement with Otto and Lewerenz (32) but in contrast with Fichtelius (12), there were fewer leukocytes situated between the epithelial cells in the jejunum of the GF rat. Although most observers have described a decrease in disaccharidase activity under the influence of cytostatics (19), the results presented here do not support this conclusion. The following reasons should be discussed regarding this discrep ancy: (1) Dose: the dose of 100mg/kg body weight is not a high dose, but suf ficient to influence the hemopoietic system (35, 45), the bladder epithelium (35) and the turnover of the intestinal epithelium (34). Perphaps disaccharidase activity is influenced only by higher doses or has recovered already at these time intervals. (2) Application: it seems unlikely that the subcutaneous application in the present investigation had an influence on the results (42). (3) Reference system: in the experiments of ae Both et al. (7) and Menge et al. (28), significant changes in villus height were not accompanied by corre sponding alterations in disaccharidase activity referred to protein. In tírese situa tions and perhaps in the present experiments, changes in enzyme activity were accompanied by similar alterations in the reference system (mucosal protein) and as a consequence of this, the specific activity of the enzyme remained unaltered. (4) Fasting: the period of fasting (48 h) is apt to change all parameters investigated in this study — for instance disaccharidase activity, protein and DNA content of the mucosa. (5) Control animals: in the present experiments, the control animals were subjected to tire same procedures as the test animals. Therefore influences of the manipulations and experimental conditions can be excluded.
The authors wish to thank W. Calvo (Abteilung für Klinische Physiologie. Universität Ulm) for preparing the histological specimens. W. Gaus (Abteilung für Medizinische Stati stik, Universität Ulm) for performing the statistical analysis, 11. Meyer (Sektion Gnoto-
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A cknowledgemen ts
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biotik, Universität Ulm) for his advice in GF technique and for confirming the GF status of the isolators, and K. Wilms (Medizinische Universitätsklinik, Tübingen) for his help in intro ducing the measurement of thymidine kinase activity. We also thank Ms. I. Schnösenberg for competent technical assistance.
References
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1 Abrams, D.G.; Bauer, H., and Sprinz, H.: Influence of the normal flora on mucosal morphology and cellular renewal in the ileum. A comparison of germfree and conven tional mice. Lab. Invest. 12: 355 364 (1963). 2 Achord, J.L.: The effect of methotrexate on rabbit intestinal mucosal enzymes and flora. Am. J. dig. Dis. 14: 315-323 (1969). 3 Altmann, G.G.: Changes in the mucosa of the smaL intestine following methotrexate administration or abdominal X-irradiation. Am. J. Anat. 140: 263-280 (1974). 4 Böhmer, R.: Rommel, K.; Goberna, R. und Raptis, S.: Disaceharidasenaktivität des Jejunums subtotal pankreatektomierter Ratten. Clinica chim. A ctaJJ: 61 -67 (1971). 5 Clarke, R.M.: Diet, mucosal architecture and epithelial cell production in the small intestine of specified-pathogen-free and conventional rats. Lab. Anim. Care 9: 201-209 (1975). 6 Dahlqvist, A.: Assay of intestinal disaccharidases. Enzymol. biol. clin. 11: 52 66 (1970). 7 De Both, N.J.; Van Dongen, J.M.; Van Hofwegen, B.; Keulemans, J.; Visser, W.J., and Galjaard, H.: The influence of various cell kinetic conditions on functional differentia tion in the small intestine of the rat. A study of enzymes bound to subcellular organ elles. J. develop. Biol. 38: 119-137 (1974). 8 Ecknauer, R. und Schnösenberg, 1.: Teilmechanisierte Bestimmung des Proteins und der Desoxyribonucleinsäure im Mikrolitermassstab in der Dünndarmschleimhaut. Z. Gastroent. (in press, 1975). 9 Eder, M.; Rostock, H. und Vogel, G.: Die Wirkung von Folsäureantagonisten (Methotrexat) auf die Regeneration der Darmschleimhaut. Virchows Arch. path. Anat. Physiol. 341: 164-176(1966). 10 Einarsson, K.; Gustafsson, J.A., and Gustafsson, B.E.: Liver microsomal hydroxylation of steroid hormones after establishing an indigenous microflora in germfree rats. Proc. Soc. exp. Biol. Med. 145: 48-52 (1974). 11 Eisenhuth, I. und Geyer, G.: Veränderungen am Dünndarmepithe! der Albinomaus durch chronische Behandlung mit den N-Iost Stoffen Endoxan, Trimitan und Degranol unter besonderer Berücksichtigung der Paneth’schen Körnerzcllcn. Acta histochem. 25: 71-85 (1966). 12 Fichtelius, K.E.: The gut epithelium a first level lymphoid organ? Expl Cell Res. 49: 87-104 (1968). 13 Fortin-Magana, R.; Hurwitz, R.; Herbst, J.J., and Kretchmer, N.: Intestinal enzymes: indicators of proliferation and differentiation in the jejunum. Science, N.Y. 167: 1627-1628 (1970). 14 Fuller, R.: The routine microbiological control of germfree isolators; in Coates The germfree animal in research (Academic Press, London 1968). 15 Galjaard. H.; Meer-Fieggen, W. van der, and Giesen, J.: Feedback control by functional villus cells on cell proliferation and maturation in intestinal epithelium. Expl Cell Res. 73: 197-207 (1972). 16 Guenet, J.L.: Sacquet, E.: Gueneau, G. et Meslin, J C.: Action de la microflora totale
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22
23
24 25 26 27 28
29
30
31 32
33
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