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The intrasanguineous host-mediated assay procedure distribution and retention of yeast in the mouse
295
Mutation Research, 64 (1979) 295--305 © Elsevier/North-Holland Biomedical Press
THE INTRASANGUINEOUS HOST-MEDIATED ASSAY PROCEDURE DISTRIBUTION AND RETENTION OF YEAST IN THE MOUSE
DOMENICO F R E Z Z A *, E R R O L ZEIGER and BHOLA N. GUPTA
Microbial Genetics Section, Laboratory of Environmental Mutagenesis and Comparative Pathology Section, Environmental Biology and Chemistry Branch, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709 (U.S.A.) (Received 13 February 1979) (Accepted 11 May 1979)
Summary A study of the factors that could affect a method to detect mutations in cells recovered from different organs after intravenous injection in mice, was performed by using the D4 strain of Saccharomyces cerevisiae. The' recovery of the yeast cells 5 min to 3 days after injection in the host animal was investigated. The circulation, distribution and localization of the cells were determined, and histopathologic analysis was performed in order to detect possible interactions between the mice and the microorganisms. We found that the yeast cells were trapped primarily in the capillaries of the organs; 3 days after injection no cells were found outside of the tissue-blood vessels. The spontaneous gene-conversion frequency of the yeast cells recovered at different times after injection was increased, but this increase was not timedependent.
The higher sensitivity of the intrasanguineous host-mediated assay (h.-m.a.) to many mutagens compared to the intraperitoneal host-mediated assay [16,17, 20] suggested that this method might be more suitable for mutagenesis studies. It is therefore necessary to investigate more accurately the various factors that may affect this new system [25]. We chose to use the eukaryotic microorganism Saccharomyces cerevisiae [7] for this study. We were interested in determining the recovery of the yeast from different organs after intravenous injection in mice. We wanted also to determine if
* Present address: L a b o r a t o r i o de Genetiea, I n s t i t u t o A n t r o p o l o g i a e P a l e o n t o l o g i a umana, Universit~ di Pisa, Via Santa Maria 53, 56100 Pisa (Italy).
296 during this time there was an increase in the spontaneous gene-conversion levels of the injected cells. The possibility that the host-immune reaction could compromise the viability of the cells was investigated histologically in order to establish if this system could be used as a tool to detect the formation of mutagenic compounds in different organs o f mammals [5,11,15]. In this study we injected the yeast S. cerevisiae D4 into mice and investigated its circulation and distribution to different organs. We recovered the injected cells from the liver, lungs, kidneys, testes and blood, and checked for efficiency of recovery and for stability of the spontaneous gene-conversion frequency. We looked at the clearance of the yeast from the blood in order to be sure that the genetic events occurring in cells recovered from a particular organ did not occur before the cells reached that organ. In addition, we determined the localization of the yeast cells in the tissues of the different organs. Materials and methods The diploid strain (D4) of Saccharomyces cerevisiae, containing two defective non-complementing alleles at the ade 2 locus (ade 2-1/ade 2-2) and at the trp 5 locus (trp 5-12/trp 5-27) [26] used as tester strain was kindly provided by Dr. David F. Callen. As complete media, YPD containing 1% Bacto-yeast extract, 2% Bacto-peptone, 2% dextrose, with or w i t h o u t 2% Bacto-agar was used. The selective media used was Bacto-yeast nitrogen base without amino acids (Difco) 0.67%, dextrose 2%, Bacto-agar 2%. It was supplemented either with 1.5% adenine sulphate or 1% L-tryptophan. The spontaneous level of gene conversion at both loci of a stationary phase culture was determined before each experiment. Male CD1 mice 7--8 weeks old were purchased from the Charles River Breeding Labs., Inc., and food (NIH-31 diet) and water were provided ad libitum. A suspension of 3 X 108 yeast cells in 0.1 ml of a physiological saline solution was injected into one of the two lateral taft veins of each mouse. At the appropriate time after the injection, the mice were anaesthetized with CO2 and a sufficient quantity of blood was recovered from the heart, then diluted and plated. To avoid coagulation, the blood was initially diluted into a 3.8% solution of sodium citrate. The volume of the blood was assumed to be 7% of the body weight of the animal. The other organs were collected aseptically and homogenized with a Potter--Elvehjem homogenizer: the liver in 6.0 ml of a 0.1 M pH 7.4 phosphate buffer; and the lungs, kidneys and testes in 4 ml o f the same solution. The final volume of each organ homogenate was recorded in order to calculate the total number of cells recovered. To determine the gene-conversion frequencies the homogenates were washed 3 times by centrifugation in a Sorvall refrigerated centrifuge at 1935 × g for 20 min so as to minimize carryover of nutrients from homogenized tissues [4]. Since cells may be lost by centrifugation, we counted the cells in the suspension with an optical microscope, and after appropriate dilution the cells were plated directly on complete media. The cells were recovered from the mice at various intervals from 5 min to 72 h after the injection. For the histological analysis, animals injected intravenously were killed by cervical dislocation 1--72 h after injection, and liver, lungs, kidneys and testes
297 were fixed in buffered isotonic neutral 10% formalin. The tissues were routinely e m b e d d e d in paraffin, cut into 6-p thick sections and stained with hematoxyline and eosine (H and E) or periodic acid Schiff (PAS) and Grotto's methenamine-silver (GMS) [ 14]. Results The cells injected into the mice were tolerated at the used dose without obvious stress. However, the presence o f extraneous particles in the blood appeared to initiate an immunological response in the lungs as indicated b y an infiltration of polymorphonuclear cells. The clearance of the yeast cells from the blood of the mouse was very rapid (Fig. 1). It was technically impossible to check the number of cells in the blood before 5 min, b u t at 5 min we had our highest rate of recovery -- 7% of the total inoculum. After 1 h less than 0.2% of the injected cells were still recoverable from the blood stream, and in 4 h in the quantity decreased to 0.04%. Thus, the cells rapidly disappeared from the peripheral circulating blood, sequestering themselves in the different organs. The location of the yeast in the various mouse organs had a direct dependence on the anatomy of the circulatory system. The microorganisms injected into the lateral tail vein will reach the right side of the heart through the vena cava and the lungs. The cells will reach the left side of the heart through the pulmonary veins and will be distributed throughout all the other organs in the animal's b o d y by arterial circulation. In agreement with the anatomy o f circulation, the lungs showed the highest recovery (30%) in the first 30 min (Fig. 1) with a slow decrease from 12% after 1 h to 1.8% after 14 h. After 3 days, it was still possible to recover 0.05% of the injected cells from the lungs. The cells can go into the liver either b y w a y of arterial circulation or through
• Liver
• 0 • 0
T .L
5_~.3~30 I Time(rain)
2
4
6 Time (hrs)
8
Lung Kidney Blood Testes
14
Fig. 1. R e c o v e r y o f viable y e a s t cells (strain D 4 of Saccharomyces cerevisiae) i n j e c t e d i n t r a v e n o u s l y in m i c e after d i f f e r e n t t i m e s o f i n c u b a t i o n f r o m d i f f e r e n t organs: liver, lungs, k i d n e y s , testes a n d b l o o d . E r r o r bars r e p r e s e n t s t a n d a r d e r r o r o f m e a n .
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Fig. 2. P h o t o m i c r o g r a p h o f t h e lung of a m o u s e 1 h a f t e r i n t r a v e n o u s i n o c u l a t i o n o f 3 X 108 y e a s t cells ( A ) N u m e r o u s d a r k - s t a i n i n g y e a s t cells are p r e s e n t in t h e capillaries o f lung a n d o n l y a f e w y e a s t cells ( a r r o w s ) are in t h e p u l m o n a r y b l o o d vessel, (B) No y e a s t cells are p r e s e n t in t h e a l v e o l a r l u m i n a . GMS stain, × 210. (C) H i g h e r m a g n i f i c a t i o n of lung s h o w i n g d a r k - s t a i n i n g y e a s t cells s t u d d e d i n t o p u l m o n a r y capillaries. PAS stain, X 520.
(Saccharomyces cerevisiae).
299 the portal system. The vena porta goes into the liver and this blood will mix with the arterial blood in the rete mirabilis. The proportion o f injected cells recovered from the liver (Fig. 1) increases after 5 rain from the injection up to the maximum of 29% obtained at 4 h. At 14 h after the injection, the recovery from this organ still exceeded 10%. After 24 h it decreased to 4% and was at 1% after 3 days. The percentage o f yeast cells recovered from the kidneys (Fig. 1) was lower than from the lungs and liver and did n o t change very much with time. The maximum level (6%) was attained after 15 min, and after 4 h 3.5% remained; after 14 h the recovery was a b o u t 1%. The number of cells recovered from the testes was not sufficient to obtain data on the spontaneous frequency o f gene conversion; recovery increased to a maximum of 0.95% at 4 h, and was 0.1% at 8 h. By means o f a histological analysis made after 1 h, it was observed that GMS stain proved to be the most desirable i n determining the approximate number and tissue distribution of yeast cells. PAS staining of tissue sections was very helpful in visualizing the phagocytosis which could n o t be visualized easily either b y GMS or H and E stains. By microscopic examination at 1 h, the lungs appeared to have the most cells per microscopic field, followed b y the kidneys and then the liver. Some o f the cells in these organs appeared in budding form. No cells could be observed in the testes or its capillaries, or in larger blood vessels. After 1 h the alveolar walls of the lung appeared to be markedly thickened due to infiltration of polymorphonuclear (PMN) cells and yeast cells. Some of the yeast cells were phagocytized by PMN cells in the lumina of the capillaries o f the alveolar walls. Very few cells were present in the larger blood vessels of the lungs (Fig. 2). In the kidneys, most of the cells were located in cortical areas, and only a few were observed in the medullary region. In the renal cortex, most of the cells were present either in the capillaries between renal tubules or in the glomerular tufts o f renal corpuscles (Fig. 3). No cells were observed either in the Bowman's capsule or in the renal tubules. The sinusoids of the liver contained most of the yeast cells (Fig. 4), some of which appeared to be phagocytized b y ,PMN cells. Tissue samples obtained 4 h after inoculation were also examined. The number o f yeast cells present in the lungs appeared to be the same as at 1 h b u t there was an increased number of PMN cells in the capillaries. The number of yeast cells present in the kidneys appeared to have declined. There were no remarkable pathological changes after 3 days that were not evident after the 4-h incubation time. The levels of gene conversion were tested at different times after the intravenous injection of the yeast cells. Table 1 shows a summary of the results obtained from the cells recovered from the liver, lungs and kidneys. The gene-conversion frequencies of cells recovered from the liver showed similar trends in the ADE and T R P loci; namely, no changes in the first 5 min, b u t significant increases with respect to control values for all times thereafter. These increases were essentially of the same magnitude at 2, 4, 8 and 14 h. The values from the kidneys were also similar; there were significant increases
Fig. 3. (A) P h o t o m i c r o g r a p h of k i d n e y of a m o u s e 1 h a f t e r i n t r a v e n o u s i n o c u l a t i o n of 3 X 108 y e a s t cells (Saccharornyces cerevisiae). N o t e t h e daxk-staining y e a s t :ells in t h e g l o m e r u l a r t u f t s , a n d n o t in t h e l a r g e r b l o o d vessel. GMS stain, X250. (B) H i g h e r m a g n i f i c a t i o n o f k i d n e y s h o w i n g t h e y e a s t cells lining t h e c a p i l l a r y vails b e t w e e n t h e r e n a l t u b u l e s . GMS stain, × 6 2 5 .
~0
TABLE 1
0.88±0.07b 0.57±0.02 0.65±0.03 0.70±0.06
0.77±0.02 0.77±0.03 0.69±0.04 0.84±0.09
0.89±0.06 0.76±0.04d 0.88±0.07c 0.90±0.08d
ADE
ADE
TRP
Liver
Control a
0.86±0.08 0.92±0.04 0.97±0.08 1.10±0.09
TRP
d d d
0.76±0.05 0.70±0.03 0.82±0.04 0.78±0.05
ADE
Lungs
c d d
CEREVIS1AE RECOVERED
0.79±0.03 0.88±0.02 0.98±0.10 0.98±0.07
TRP
d c c
FROM DIFFERENT
T h e level o f t h e g e n e c o n v e r s i o n in t h e c o n t r o l w a s o b t a i n e d f r o m t h e cell s u s p e n s i o n a t t h e t i m e o f i n j e c t i o n i n t o t h e m o u s e . C o n v e r t a n t s p e r 10 $ cells ± S.E. p <~ 0 . 0 5 . p <~ 0 . 0 1 ( p a i r e d t t e s t ) .
8 9 8 9
5mm 4h 8h 14h
a b c d
Number of animals
Time
SUMMARY OF THE VALUES OF GENE CONVERSION IN SACCHAROMYCES TION IN MICE WITH THE INTRASANGUINEOUS HOST-MEDIATED ASSAY
1.32±0.13 0.92±0.07 0.94±0.10 1.18±0.11
ADE
Kidneys
c d d d
1.59±0.18 1.17±0.09 1.38±0.11 1.46±0.12
TRP d d d d
ORGANS AFTER INCUBA-
302
Fig. 4. P h o t o m i c r o g r a p h o f liver of a m o u s e 1 h a f t e r i n t r a v e n o u s i n o c u l a t i o n o f 3 × 108 y e a s t cells
(Saccharomyces cerevisiae). N o t e t h e d a r k - s t a i n i n g y e a s t cells in t h e h e p a t i c sinusoids. PAS stain, X 6 2 5 .
over control data at all observed times. Increases seen at 2 h were much greater than the increases seen at any other time. However, in the initial experiments performed at this time the cells were washed using a less effective procedure. Therefore, these results cannot be directly compared with those at other times and are not presented in the table. In the lungs, gene conversion frequency had a significant increase at the ADE locus at 2, 4 and 8 h. The TRP locus showed no effect at 5 min and at 2 h, but there were significant increases with respect to control levels at 4, 8 and 14 h.
Discussion One of the problems that had to be solved in the use of the intrasanguineous h.-m.a, was the localization of the microorganisms in the organs of the host animal. First, we found how Saccharomyces cerevisiae cells were distributed in the body of the host with time, and secondly, by using histological techniques, we determined the localization of the microorganisms in the tissues inside the various organs. The intrasanguineous host-mediated assay is preferred over the intraperitoneal host-mediated assay because it provides the most direct contact of the mutagen with the indicator microorganism. The system seems to be very sensitive, but it is important to know with which tissues the indicator cells are in close contact [20]. As suggested by several authors, the possibility that the indicator microorganisms are inside the interstitial space or, better yet, inside the cells of the
303
tissues, could make the system much more sensitive [7,12,21]. Although we did n o t find any yeast cells outside the blood stream, the localization of these celis in the capillaries of the different organs is sufficient to increase the sensitivity o f the test above that found for the intraperitoneal h.-m.a. [2,10]. The histological analysis o f the organs of animals treated b y intravenous injection of S. cerevisiae showed that the location inside the tissue was similar for each organ. Most of the yeast cells were trapped in the capillaries of the lungs, kidneys and liver. This could be one o f the reasons for the low recovery of microorganisms from the heart blood. No yeast cells could be observed in the testes at microscopic examination. This was n o t unexpected since the number of microorganisms that can be recovered from the testes is very low when compared with lungs, kidneys and liver. Testes contain very few capillaries compared to the other three organs, are functionally different, and have a lower internal temperature. Moreover, the other organs (lungs, kidneys, liver) are more involved in the elimination of extraneous organisms. It appeared that when given intravenously the cells remained in the blood vessels, primarily in the capillaries, or were phagocytized by PMN. The concentration of the yeast cells in the cortical area of the kidneys is rather helpful in studying in the mutagenesis of c o m p o u n d s requiring metabolic activation by the mixed-funtion oxidase system, which is a b o u t three times more active in the cortex than in the medullary region [ 13]. The population o f injected cells in the organs changes with time. Based upon h a e m o c y t o m e t e r counts of the yeast cells in the organ homogenates prior to plating it can be presumed that non-viable cells did n o t constitute a significant fraction of the population. The apparent disagreement between the density of the yeast in the various organs found with histological observation and the recovery obtained b y homogenizing the organs and plating the cells, can be resolved by dividing the number of cells recovered b y the organ weight. After 1 h of incubation, the quantity of cells per gram of organ is: 22 × 106 for the liver; 96.1 × 106 for the lungs, and 20.6 × 106 for the kidneys. It is not known, however, if the decreases in recovered cells are due to their being sequestered in organs other than those examined in this study or to their being killed in situ b y the polymorphonuclear cells. The clearance of the yeasts from the circulating blood is very fast since they are trapped in the capillaries of the various organs. This supports the hypothesis that the majority o f the yeast cells are n o t circulating after less than 1 h. The experiments were performed so as to affect the mouse as little as possible. After injecting the mice with different volumes and concentrations of cells, 3 × 108 cells was chosen as a cell concentration that was not toxic, b u t which allowed sufficient recovery o f cells to determine a spontaneous level of gene conversion at the various time points. Although the best recovery in the intrasanguineous host-mediated assay was found with eucaryotic organisms, it is possible to use the SV3, T A 1 0 0 and TA98 strains o f Salmonella typhimurium [1,10], some strains of Escherichia coli [20] or bacteriophages [22], all of which can be recovered from various organs. However, only the yeast and the Salmonella SV3 can be recovered from the mice after a prolonged incubation period. A small b u t constant increase over the control was observed in the levels o f
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gene conversion in the cells recovered from the organs at all times up to 14 h after injection. The large increase over the control at the TRP locus of the cells recovered from the kidneys was also reported from other similar experiments [2]. The presence of catabolites of protein in addition to tissue c o m p o n e n t s in the kidneys of the mouse, might enhance the level of the gene conversion at the t w o loci. A potential problem with this system is that the immune response to the injected cells may alter the physiological equilibrium of the host organism. Up to the present time, n o b o d y has reported on the use of the intrasanguineous host-mediated assay for long-term exposure [18]. Chemicals which are not detected as mutagens at acute, b u t toxic, doses may have significant mutagenic activity with chronic exposure of the animal to the c o m p o u n d . Obviously, this is one of the advantages of working with the whole animal. Moreover, chemicals that are difficult to handle (volatile, insoluble, unstable, etc.) and chemicals having u n k n o w n metabolic pathways, or for which the enzymes present in the extract of the single organs are not sufficient to permit metabolic transformation, can be studied more effectively within the whole animal than b y using an organ extract in vitro [19]. The high efficiency of recovery of indicator cells from liver, lungs and kidneys makes this system very useful, since it is possible to run experiments with a long incubation time, which might be necessary with c o m p o u n d s which require a long time for complete absorption and metabolism. It has been shown that the response of different organs to chemical mutagens can be significantly different [2,6,10,24]. This can be an important consideration when it is of interest to assay chemicals in a short-term mutagenesis test, in order to see if there is a specific target organ for the c o m p o u n d or its metabolites. In mammals the organs from which we are recovering the cells are metabolically the most active. Finally, since enzymatic activities in different organs vary with species and strains of mammals, and are related to sex, age and diet [3,8,9,19,24,25] the intrasanguineous host-mediated assay may be a valuable tool in studying these metabolic differences in vivo.
Acknowledgements The authors thank Ruth Sorg, Clyde Moore and Patrick Pitts for their excellent technical assistance.
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M a t t e r , L o c a l i z a t i o n o f E. coli K - 1 2 in livers o f m i c e u s e d f o r a n i n t r a s a n g u i n e o u s host-mediated assay, Mutation Res., 46 (1977) 45--48. 1 3 I c h i h a r a , K . , E. K u s u n o s e a n d M. K u s u n o s e , S o m e p r o p e r t i e s a n d d i s t r i b u t i o n o f t h e o . ~ - h y d r o x y l a t i o n system of medium-chain fatty acids, Biochim. Biophys. Acta, 176 (1969) 704--712. 14 Luna, L.G., Manual of histologic staining methods of the Armed Forces Institute of Pathology, M c G r a w Hill, N e w Y o r k , 1 9 6 8 . 1 5 M a g e e , P . N . , a n d J . M . B a r n e s , T h e p r o d u c t i o n o f m a l i g n a n t p r i m a r y h e p a t i c t u m o r s in t h e r a t b y f e e d i n g d i m e t h y l n i t r o s a m i n e , Br. J. C a n c e r , I 0 ( 1 9 5 6 ) 1 1 4 - - 1 2 1 . 1 6 Mailing, H . V . , C o m p a r i s o n b e t w e e n in v i t r o a n d in vivo a c t i v a t i o n : i n : F . J . De Serres et al. ( E d s . ) , In v i t r o m e t a b o l i c a c t i v a t i o n in m u t a g e n e s i s t e s t i n g , N o r t h - H o l l a n d , A m s t e r d a m , 1 9 7 6 , p p . 1 4 3 - - 1 5 5 . 1 7 Mailing, H . V . , M u t a g e n i c a c t i v a t i o n o f d i m e t h y l n i t r o s a m i n e a n d d i e t h y l n i t r o s a m i n e in t h e h o s t - m e d i ated assay and the microsomal system, Mutation Res., 26 (1974) 485--472. 1 8 Mailing, H . V . , M u t a t i o n i n d u c t i o n in N e u r o s p o r a crassa i n c u b a t e d in m i c e a n d r a t s , Mol. G e n . G e n e t . , 116 (1972) 211--222. 19 Mailing, H . V . , a n d C . N . F r a n t z , In v i t r o v e r s u s in vivo m e t a b o l i c a c t i v a t i o n o f n i u t a g e n s , E n v i r o n . Health Perspeet., 6 (1973) 71--83. 2 0 M o h n , G., a n d J. E l l e n b e r g e r , M a m m a l i a n b l o o d m u t a g e n i c i t y t e s t s u s i n g a m u l t i p u r p o s e s t r a i n o f E s c h e r i c h i a coli K - 1 2 , M u t a t i o n R e s . , 19 ( 1 9 7 3 ) 2 5 7 - - 2 6 0 . 21 M o h n , G., J . E l l e n b e r g e r , D. M c G r e g o r a n d M. H a n s - J o a c h i m , M u t a g e n i c i t y s t u d i e s in m i c r o o r g a n i s m s in v i t r o , w i t h e x t r a c t s o f m a m m a l i a n o r g a n s a n d w i t h t h e h o s t - m e d i a t e d a s s a y , M u t a t i o n R e s . , 2 9 (1975) 221--233. 2 2 M o h n , G., P. M a i e r a n d K . Biirki, T h e use o f b a c t e r i o p h a g e s as i n d i c a t o r s in h o s t - m e d i a t e d a s s a y , M u t a t i o n Res., 4 8 ( 1 9 7 7 ) 3 6 7 - - 3 7 0 . 2 3 P r o p p i n g , P., a n d W. B u s e l m a l e r , T h e i n f l u e n c e o f m e t a b o l i s m o n m u t a g e n i c a c t i v i t y in t h e h o s t - m e d i ated assay, Arch. Toxicol., 28 (1971) 129--134. 2 4 W h o n g , W.-Z., a n d T.M. O n g , C o m p a r a t i v e s t u d i e s o n t h e m e d i a t e d m u t a g e n i c i t y o f d i m e t h y l n i t r o s a m i n e i n N e u r o s p o r a crassa, 9 t h A n n u . M e e t i n g A m . E n v i r o n . M u t a g e n S o c . , S a n F r a n c i s c o , C A , M a r c h 9--13, 1978. 2 5 Z e i g e r , E., D i e t a r y m o d i f i c a t i o n s a f f e c t i n g t h e m u t a g e n i c i t y o f N - n i t r o s o c o m p o u n d s in t h e h o s t mediated assay response, Cancer Res., 35 (1975) 1813--1818. 26 Z i m m e r m a n , F . K . , I n d u c t i o n o f m i t o t i c g e n e c o n v e r s i o n b y m u t a g e n s , M u t a t i o n R e s . , 11 ( 1 9 7 1 ) 327--337.
Mutation Research, 64 (1979) 295--305 © Elsevier/North-Holland Biomedical Press
THE INTRASANGUINEOUS HOST-MEDIATED ASSAY PROCEDURE DISTRIBUTION AND RETENTION OF YEAST IN THE MOUSE
DOMENICO F R E Z Z A *, E R R O L ZEIGER and BHOLA N. GUPTA
Microbial Genetics Section, Laboratory of Environmental Mutagenesis and Comparative Pathology Section, Environmental Biology and Chemistry Branch, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709 (U.S.A.) (Received 13 February 1979) (Accepted 11 May 1979)
Summary A study of the factors that could affect a method to detect mutations in cells recovered from different organs after intravenous injection in mice, was performed by using the D4 strain of Saccharomyces cerevisiae. The' recovery of the yeast cells 5 min to 3 days after injection in the host animal was investigated. The circulation, distribution and localization of the cells were determined, and histopathologic analysis was performed in order to detect possible interactions between the mice and the microorganisms. We found that the yeast cells were trapped primarily in the capillaries of the organs; 3 days after injection no cells were found outside of the tissue-blood vessels. The spontaneous gene-conversion frequency of the yeast cells recovered at different times after injection was increased, but this increase was not timedependent.
The higher sensitivity of the intrasanguineous host-mediated assay (h.-m.a.) to many mutagens compared to the intraperitoneal host-mediated assay [16,17, 20] suggested that this method might be more suitable for mutagenesis studies. It is therefore necessary to investigate more accurately the various factors that may affect this new system [25]. We chose to use the eukaryotic microorganism Saccharomyces cerevisiae [7] for this study. We were interested in determining the recovery of the yeast from different organs after intravenous injection in mice. We wanted also to determine if
* Present address: L a b o r a t o r i o de Genetiea, I n s t i t u t o A n t r o p o l o g i a e P a l e o n t o l o g i a umana, Universit~ di Pisa, Via Santa Maria 53, 56100 Pisa (Italy).
296 during this time there was an increase in the spontaneous gene-conversion levels of the injected cells. The possibility that the host-immune reaction could compromise the viability of the cells was investigated histologically in order to establish if this system could be used as a tool to detect the formation of mutagenic compounds in different organs o f mammals [5,11,15]. In this study we injected the yeast S. cerevisiae D4 into mice and investigated its circulation and distribution to different organs. We recovered the injected cells from the liver, lungs, kidneys, testes and blood, and checked for efficiency of recovery and for stability of the spontaneous gene-conversion frequency. We looked at the clearance of the yeast from the blood in order to be sure that the genetic events occurring in cells recovered from a particular organ did not occur before the cells reached that organ. In addition, we determined the localization of the yeast cells in the tissues of the different organs. Materials and methods The diploid strain (D4) of Saccharomyces cerevisiae, containing two defective non-complementing alleles at the ade 2 locus (ade 2-1/ade 2-2) and at the trp 5 locus (trp 5-12/trp 5-27) [26] used as tester strain was kindly provided by Dr. David F. Callen. As complete media, YPD containing 1% Bacto-yeast extract, 2% Bacto-peptone, 2% dextrose, with or w i t h o u t 2% Bacto-agar was used. The selective media used was Bacto-yeast nitrogen base without amino acids (Difco) 0.67%, dextrose 2%, Bacto-agar 2%. It was supplemented either with 1.5% adenine sulphate or 1% L-tryptophan. The spontaneous level of gene conversion at both loci of a stationary phase culture was determined before each experiment. Male CD1 mice 7--8 weeks old were purchased from the Charles River Breeding Labs., Inc., and food (NIH-31 diet) and water were provided ad libitum. A suspension of 3 X 108 yeast cells in 0.1 ml of a physiological saline solution was injected into one of the two lateral taft veins of each mouse. At the appropriate time after the injection, the mice were anaesthetized with CO2 and a sufficient quantity of blood was recovered from the heart, then diluted and plated. To avoid coagulation, the blood was initially diluted into a 3.8% solution of sodium citrate. The volume of the blood was assumed to be 7% of the body weight of the animal. The other organs were collected aseptically and homogenized with a Potter--Elvehjem homogenizer: the liver in 6.0 ml of a 0.1 M pH 7.4 phosphate buffer; and the lungs, kidneys and testes in 4 ml o f the same solution. The final volume of each organ homogenate was recorded in order to calculate the total number of cells recovered. To determine the gene-conversion frequencies the homogenates were washed 3 times by centrifugation in a Sorvall refrigerated centrifuge at 1935 × g for 20 min so as to minimize carryover of nutrients from homogenized tissues [4]. Since cells may be lost by centrifugation, we counted the cells in the suspension with an optical microscope, and after appropriate dilution the cells were plated directly on complete media. The cells were recovered from the mice at various intervals from 5 min to 72 h after the injection. For the histological analysis, animals injected intravenously were killed by cervical dislocation 1--72 h after injection, and liver, lungs, kidneys and testes
297 were fixed in buffered isotonic neutral 10% formalin. The tissues were routinely e m b e d d e d in paraffin, cut into 6-p thick sections and stained with hematoxyline and eosine (H and E) or periodic acid Schiff (PAS) and Grotto's methenamine-silver (GMS) [ 14]. Results The cells injected into the mice were tolerated at the used dose without obvious stress. However, the presence o f extraneous particles in the blood appeared to initiate an immunological response in the lungs as indicated b y an infiltration of polymorphonuclear cells. The clearance of the yeast cells from the blood of the mouse was very rapid (Fig. 1). It was technically impossible to check the number of cells in the blood before 5 min, b u t at 5 min we had our highest rate of recovery -- 7% of the total inoculum. After 1 h less than 0.2% of the injected cells were still recoverable from the blood stream, and in 4 h in the quantity decreased to 0.04%. Thus, the cells rapidly disappeared from the peripheral circulating blood, sequestering themselves in the different organs. The location of the yeast in the various mouse organs had a direct dependence on the anatomy of the circulatory system. The microorganisms injected into the lateral tail vein will reach the right side of the heart through the vena cava and the lungs. The cells will reach the left side of the heart through the pulmonary veins and will be distributed throughout all the other organs in the animal's b o d y by arterial circulation. In agreement with the anatomy o f circulation, the lungs showed the highest recovery (30%) in the first 30 min (Fig. 1) with a slow decrease from 12% after 1 h to 1.8% after 14 h. After 3 days, it was still possible to recover 0.05% of the injected cells from the lungs. The cells can go into the liver either b y w a y of arterial circulation or through
• Liver
• 0 • 0
T .L
5_~.3~30 I Time(rain)
2
4
6 Time (hrs)
8
Lung Kidney Blood Testes
14
Fig. 1. R e c o v e r y o f viable y e a s t cells (strain D 4 of Saccharomyces cerevisiae) i n j e c t e d i n t r a v e n o u s l y in m i c e after d i f f e r e n t t i m e s o f i n c u b a t i o n f r o m d i f f e r e n t organs: liver, lungs, k i d n e y s , testes a n d b l o o d . E r r o r bars r e p r e s e n t s t a n d a r d e r r o r o f m e a n .
298
Fig. 2. P h o t o m i c r o g r a p h o f t h e lung of a m o u s e 1 h a f t e r i n t r a v e n o u s i n o c u l a t i o n o f 3 X 108 y e a s t cells ( A ) N u m e r o u s d a r k - s t a i n i n g y e a s t cells are p r e s e n t in t h e capillaries o f lung a n d o n l y a f e w y e a s t cells ( a r r o w s ) are in t h e p u l m o n a r y b l o o d vessel, (B) No y e a s t cells are p r e s e n t in t h e a l v e o l a r l u m i n a . GMS stain, × 210. (C) H i g h e r m a g n i f i c a t i o n of lung s h o w i n g d a r k - s t a i n i n g y e a s t cells s t u d d e d i n t o p u l m o n a r y capillaries. PAS stain, X 520.
(Saccharomyces cerevisiae).
299 the portal system. The vena porta goes into the liver and this blood will mix with the arterial blood in the rete mirabilis. The proportion o f injected cells recovered from the liver (Fig. 1) increases after 5 rain from the injection up to the maximum of 29% obtained at 4 h. At 14 h after the injection, the recovery from this organ still exceeded 10%. After 24 h it decreased to 4% and was at 1% after 3 days. The percentage o f yeast cells recovered from the kidneys (Fig. 1) was lower than from the lungs and liver and did n o t change very much with time. The maximum level (6%) was attained after 15 min, and after 4 h 3.5% remained; after 14 h the recovery was a b o u t 1%. The number of cells recovered from the testes was not sufficient to obtain data on the spontaneous frequency o f gene conversion; recovery increased to a maximum of 0.95% at 4 h, and was 0.1% at 8 h. By means o f a histological analysis made after 1 h, it was observed that GMS stain proved to be the most desirable i n determining the approximate number and tissue distribution of yeast cells. PAS staining of tissue sections was very helpful in visualizing the phagocytosis which could n o t be visualized easily either b y GMS or H and E stains. By microscopic examination at 1 h, the lungs appeared to have the most cells per microscopic field, followed b y the kidneys and then the liver. Some o f the cells in these organs appeared in budding form. No cells could be observed in the testes or its capillaries, or in larger blood vessels. After 1 h the alveolar walls of the lung appeared to be markedly thickened due to infiltration of polymorphonuclear (PMN) cells and yeast cells. Some of the yeast cells were phagocytized by PMN cells in the lumina of the capillaries o f the alveolar walls. Very few cells were present in the larger blood vessels of the lungs (Fig. 2). In the kidneys, most of the cells were located in cortical areas, and only a few were observed in the medullary region. In the renal cortex, most of the cells were present either in the capillaries between renal tubules or in the glomerular tufts o f renal corpuscles (Fig. 3). No cells were observed either in the Bowman's capsule or in the renal tubules. The sinusoids of the liver contained most of the yeast cells (Fig. 4), some of which appeared to be phagocytized b y ,PMN cells. Tissue samples obtained 4 h after inoculation were also examined. The number o f yeast cells present in the lungs appeared to be the same as at 1 h b u t there was an increased number of PMN cells in the capillaries. The number of yeast cells present in the kidneys appeared to have declined. There were no remarkable pathological changes after 3 days that were not evident after the 4-h incubation time. The levels of gene conversion were tested at different times after the intravenous injection of the yeast cells. Table 1 shows a summary of the results obtained from the cells recovered from the liver, lungs and kidneys. The gene-conversion frequencies of cells recovered from the liver showed similar trends in the ADE and T R P loci; namely, no changes in the first 5 min, b u t significant increases with respect to control values for all times thereafter. These increases were essentially of the same magnitude at 2, 4, 8 and 14 h. The values from the kidneys were also similar; there were significant increases
Fig. 3. (A) P h o t o m i c r o g r a p h of k i d n e y of a m o u s e 1 h a f t e r i n t r a v e n o u s i n o c u l a t i o n of 3 X 108 y e a s t cells (Saccharornyces cerevisiae). N o t e t h e daxk-staining y e a s t :ells in t h e g l o m e r u l a r t u f t s , a n d n o t in t h e l a r g e r b l o o d vessel. GMS stain, X250. (B) H i g h e r m a g n i f i c a t i o n o f k i d n e y s h o w i n g t h e y e a s t cells lining t h e c a p i l l a r y vails b e t w e e n t h e r e n a l t u b u l e s . GMS stain, × 6 2 5 .
~0
TABLE 1
0.88±0.07b 0.57±0.02 0.65±0.03 0.70±0.06
0.77±0.02 0.77±0.03 0.69±0.04 0.84±0.09
0.89±0.06 0.76±0.04d 0.88±0.07c 0.90±0.08d
ADE
ADE
TRP
Liver
Control a
0.86±0.08 0.92±0.04 0.97±0.08 1.10±0.09
TRP
d d d
0.76±0.05 0.70±0.03 0.82±0.04 0.78±0.05
ADE
Lungs
c d d
CEREVIS1AE RECOVERED
0.79±0.03 0.88±0.02 0.98±0.10 0.98±0.07
TRP
d c c
FROM DIFFERENT
T h e level o f t h e g e n e c o n v e r s i o n in t h e c o n t r o l w a s o b t a i n e d f r o m t h e cell s u s p e n s i o n a t t h e t i m e o f i n j e c t i o n i n t o t h e m o u s e . C o n v e r t a n t s p e r 10 $ cells ± S.E. p <~ 0 . 0 5 . p <~ 0 . 0 1 ( p a i r e d t t e s t ) .
8 9 8 9
5mm 4h 8h 14h
a b c d
Number of animals
Time
SUMMARY OF THE VALUES OF GENE CONVERSION IN SACCHAROMYCES TION IN MICE WITH THE INTRASANGUINEOUS HOST-MEDIATED ASSAY
1.32±0.13 0.92±0.07 0.94±0.10 1.18±0.11
ADE
Kidneys
c d d d
1.59±0.18 1.17±0.09 1.38±0.11 1.46±0.12
TRP d d d d
ORGANS AFTER INCUBA-
302
Fig. 4. P h o t o m i c r o g r a p h o f liver of a m o u s e 1 h a f t e r i n t r a v e n o u s i n o c u l a t i o n o f 3 × 108 y e a s t cells
(Saccharomyces cerevisiae). N o t e t h e d a r k - s t a i n i n g y e a s t cells in t h e h e p a t i c sinusoids. PAS stain, X 6 2 5 .
over control data at all observed times. Increases seen at 2 h were much greater than the increases seen at any other time. However, in the initial experiments performed at this time the cells were washed using a less effective procedure. Therefore, these results cannot be directly compared with those at other times and are not presented in the table. In the lungs, gene conversion frequency had a significant increase at the ADE locus at 2, 4 and 8 h. The TRP locus showed no effect at 5 min and at 2 h, but there were significant increases with respect to control levels at 4, 8 and 14 h.
Discussion One of the problems that had to be solved in the use of the intrasanguineous h.-m.a, was the localization of the microorganisms in the organs of the host animal. First, we found how Saccharomyces cerevisiae cells were distributed in the body of the host with time, and secondly, by using histological techniques, we determined the localization of the microorganisms in the tissues inside the various organs. The intrasanguineous host-mediated assay is preferred over the intraperitoneal host-mediated assay because it provides the most direct contact of the mutagen with the indicator microorganism. The system seems to be very sensitive, but it is important to know with which tissues the indicator cells are in close contact [20]. As suggested by several authors, the possibility that the indicator microorganisms are inside the interstitial space or, better yet, inside the cells of the
303
tissues, could make the system much more sensitive [7,12,21]. Although we did n o t find any yeast cells outside the blood stream, the localization of these celis in the capillaries of the different organs is sufficient to increase the sensitivity o f the test above that found for the intraperitoneal h.-m.a. [2,10]. The histological analysis o f the organs of animals treated b y intravenous injection of S. cerevisiae showed that the location inside the tissue was similar for each organ. Most of the yeast cells were trapped in the capillaries of the lungs, kidneys and liver. This could be one o f the reasons for the low recovery of microorganisms from the heart blood. No yeast cells could be observed in the testes at microscopic examination. This was n o t unexpected since the number of microorganisms that can be recovered from the testes is very low when compared with lungs, kidneys and liver. Testes contain very few capillaries compared to the other three organs, are functionally different, and have a lower internal temperature. Moreover, the other organs (lungs, kidneys, liver) are more involved in the elimination of extraneous organisms. It appeared that when given intravenously the cells remained in the blood vessels, primarily in the capillaries, or were phagocytized by PMN. The concentration of the yeast cells in the cortical area of the kidneys is rather helpful in studying in the mutagenesis of c o m p o u n d s requiring metabolic activation by the mixed-funtion oxidase system, which is a b o u t three times more active in the cortex than in the medullary region [ 13]. The population o f injected cells in the organs changes with time. Based upon h a e m o c y t o m e t e r counts of the yeast cells in the organ homogenates prior to plating it can be presumed that non-viable cells did n o t constitute a significant fraction of the population. The apparent disagreement between the density of the yeast in the various organs found with histological observation and the recovery obtained b y homogenizing the organs and plating the cells, can be resolved by dividing the number of cells recovered b y the organ weight. After 1 h of incubation, the quantity of cells per gram of organ is: 22 × 106 for the liver; 96.1 × 106 for the lungs, and 20.6 × 106 for the kidneys. It is not known, however, if the decreases in recovered cells are due to their being sequestered in organs other than those examined in this study or to their being killed in situ b y the polymorphonuclear cells. The clearance of the yeasts from the circulating blood is very fast since they are trapped in the capillaries of the various organs. This supports the hypothesis that the majority o f the yeast cells are n o t circulating after less than 1 h. The experiments were performed so as to affect the mouse as little as possible. After injecting the mice with different volumes and concentrations of cells, 3 × 108 cells was chosen as a cell concentration that was not toxic, b u t which allowed sufficient recovery o f cells to determine a spontaneous level of gene conversion at the various time points. Although the best recovery in the intrasanguineous host-mediated assay was found with eucaryotic organisms, it is possible to use the SV3, T A 1 0 0 and TA98 strains o f Salmonella typhimurium [1,10], some strains of Escherichia coli [20] or bacteriophages [22], all of which can be recovered from various organs. However, only the yeast and the Salmonella SV3 can be recovered from the mice after a prolonged incubation period. A small b u t constant increase over the control was observed in the levels o f
304
gene conversion in the cells recovered from the organs at all times up to 14 h after injection. The large increase over the control at the TRP locus of the cells recovered from the kidneys was also reported from other similar experiments [2]. The presence of catabolites of protein in addition to tissue c o m p o n e n t s in the kidneys of the mouse, might enhance the level of the gene conversion at the t w o loci. A potential problem with this system is that the immune response to the injected cells may alter the physiological equilibrium of the host organism. Up to the present time, n o b o d y has reported on the use of the intrasanguineous host-mediated assay for long-term exposure [18]. Chemicals which are not detected as mutagens at acute, b u t toxic, doses may have significant mutagenic activity with chronic exposure of the animal to the c o m p o u n d . Obviously, this is one of the advantages of working with the whole animal. Moreover, chemicals that are difficult to handle (volatile, insoluble, unstable, etc.) and chemicals having u n k n o w n metabolic pathways, or for which the enzymes present in the extract of the single organs are not sufficient to permit metabolic transformation, can be studied more effectively within the whole animal than b y using an organ extract in vitro [19]. The high efficiency of recovery of indicator cells from liver, lungs and kidneys makes this system very useful, since it is possible to run experiments with a long incubation time, which might be necessary with c o m p o u n d s which require a long time for complete absorption and metabolism. It has been shown that the response of different organs to chemical mutagens can be significantly different [2,6,10,24]. This can be an important consideration when it is of interest to assay chemicals in a short-term mutagenesis test, in order to see if there is a specific target organ for the c o m p o u n d or its metabolites. In mammals the organs from which we are recovering the cells are metabolically the most active. Finally, since enzymatic activities in different organs vary with species and strains of mammals, and are related to sex, age and diet [3,8,9,19,24,25] the intrasanguineous host-mediated assay may be a valuable tool in studying these metabolic differences in vivo.
Acknowledgements The authors thank Ruth Sorg, Clyde Moore and Patrick Pitts for their excellent technical assistance.
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