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Cytotoxin from Pseudomonas aeruginosa in mice: Distribution related to pathology
To~ Vol 19, No. 6. pp. 763-771,1981 . Printedin Great &ihm.
0041-MOl/81f6076}-09 m~ O 1981 Perpmm Prey Ltd
CYTOTOXIN FROM PSEUDOMONAS AERUGINOSA IN MICE : DISTRIBUTION RELATED TO PATHOLOGY* F. LUTzt$, S. G~BERt and I. KXUFm§ t Institute of Pharmacology and Toxicology, and §Institute of Veterinary Pathology, Justus-Liebig-University, D-63 Giessen, Federal Republic of Germany (Accepted for publication 12 May 1981)
F. Lurz, SGmPsHAat;Rand 1. KXuFmt. Cytotoxin from Pseudomonaa aengbeosa in mice : distribution related to pathology. Taxiton 19, 763-771,1981 .-The cytotoxin from Pseudomonas aeruginow was labelled with iodine using the lactoperoxidaw/glucose oxidasereaction The relationship between the degree of iodination andcell toxicity wasdetermined . Afteri.v.application of sublethal doses (0 .03leg) of the [I I'I]-cytotoxin (specific activity 311&i/,ug) into mice, 90/of the radioactivity was taken up and distributed among all organs inve ted except the brain. The cytotoxin concentration, calculated on the basis of protein-bound [12 1], washighestin theurine. Lethal i.v. doses(2 pg) of the cytotoxin induced dehydratiomkHistological examination showed degeneration of epithelial cells in the distal kidney tubuli in addition to necrosis of liver lobuli. The damage to kidney tubuli seemsto be responsible for the toxicity of the cytotoxin in truce. INTRODUCTION
A cyTOToxic protein with a molecular weight of25,100 daltons, produced by Pseudomonas aeruginosa (originally isolated from bovine milky is toxic to nearly all mammalian cells (SCHARMANN,.1976b ; FRIMMI?R et A, 1976b ; LUTz,1979~ In studieson isolated cells a dosedependent increase ofcell volume and potassium loss was observed (FRIMME1t et al., 1976b~ However, the mechanism by which the toxin acts is not yet clear. MATERIALS AND METHODS The cytotoxin was prepared from an autolysate of Pseudomonat aerugblosa (LuTz, 1979} Iodination was performed enzymatically according to the method of MtYACHI et al. (19731 with immobilized lactoperoxidase/glucose oxidas (Fazymobead®, Biorad, Mifnchen, FRG} The cytotoxm (50pg) was reacted with unlabelled sodium iodide containing about 0.03 yes (1 kBq) of [1 ='1] (carrier free, Amersham-Buchbr, Braunschweig, FRG) in a total volume of 701d and at molarratios of toxin : iodide from 1 :1 to 1 :10. After various time intervals aliquots of the incubation mixture were tested for precipitable radioactivity using picric acid. The remaining mixture was centrifuged andthesupernatant wastested for toxicity againstbutnan granulocytes in aslide adhesion test (ScHARmANN, 1976x). Seventy per ant of the radioactivity was precipitable within 5 min. The biological activity of the cytotoxin was affected both by the duration of the labelling and the amount of coupled iodine (Fig 1). At one and two moles of iodine per mole of the toxin a loss of its toxicity could not be detected after 5 minof iodination . For thedistribution studies in vivo the cytotoxin wasiodinated with Na[11'I] at aratio of 1 : 1.3 for5 min. To increase theprecipitability to 96-99'/theeluate of the [1:'I]-cytotoxin fraction from a Sephadex G 25 column was dialyzed againstphosphate-buffered aaline. A specific radioactivity of31 f 9pCi/Ng (1 .1 MBq/pg) from nine experiments was calculated. The labelled protein co-migrated with the unlabelled cytotomn in gel electrophoresisand could be stored forfew days at 4°G At thebeginningof each distribution studyprecipitability of the radioactivity of the ["'I]- ytotoxin was controlled. The charge was removed if precipitability was lowerthan 96'/, * A preliminary report has been ptaetlted at the 21st Spring Meeting of the German Pharmacological Society, Mainz 1980 and published in abstract form in Naunyn-Schmiedeberg's Arch. Pharenacol. 1980, 311, suppl, R28. $ Address for corraspondenee. 763
F. LUTZ, S. GRIESHABER and I. KÄUFER
764
Precipitable radioactivity
1001
100
'/.
Relative toxicity against 50 granulocytes
50
0
0 0
5 10 min Iodination time
15
FIG. 1. TIME DEPENDENCE OF THE IODINE LABELLING OF THE CYTOTOXIN AND ITS TOXIC A(TIvrry .
The iodination was performed by the lactoperoxidaw/glucose oxidase reaction. The iodination mixture containeda ratioof toxin : iodide of 1 :1 with traces of [I=sI] . The toxicity ofthecytotoxin for human granulocytes wasdetermined in a slideadhesion test 0---A, radioactivity precipitable with all picric acid ; 0-", 50'/ of the granulocytes were swollen within 30min ; 0-0, granulocytes were swollen within 30 min.
Protein binding of radioactivity was determined by precipitation with equal amounts of saturated picric acid in the presence of bovine serum albumin. In the case of serum, the reaction of toxin with rabbit antitoxin was also utilized . Tests were performed with antitoxin in solution or coupled to protein A-positive Staphylococci (KrrzRow et al., 1979). If the antitoxin in solution was used, non-labelled cytotoxin was added to the incubation vessel at the end of the 1 hr incubation to increase the total sediment After washing the sediment the radioactivity was determined. A Packard Autogammata counter (efficiency 68%) was used to measure the radioactivity. Protein was determined by the method of LOWRY et al. (1951 Sodium dodecyl sulphate polyacrylamide gel electrophoresis was performed as described by SWANK and MUNKRES (1971 Antitoxin was prepared as described by HARBOE and INOILD (1973). Female HAN-NMRI mice of about30 gbody weight,bred in ourfacilities,were used . Fordistribution studies, the drinking water contained 0.42'/ NaCl and 0.05% NaI and wasprovided ad fiNtwn for two weeks. Urine was collected by means of polyethylene-ooated filter paper on the cage floor. The mice were anesthetized with ether before removal of the organs . The radioactivity data of serum were analyzed by the method of least squares, using the linear function :
(1) In (y) = In (a) + (b t sr) - t t %( y).., where y is the quotient of cpm found per g serum and cpm injected per g body weight The terms in (a) and b are parameters of the function, t is the time after injection of radioactivity, sb is the standard deviation of b and 3Wy).x Is the standard deviation of the function . The half-time is given by 1/b - In(2} The remaining statistical analysis was carried out using the method of SACHS (1978). All standard deviations except sr are standard deviations of single values. RESULTS
Distribution of radioactivity after application of small amounts of [ 125 1]-eytotoxin (about 3 x 10 a g) or free [1231] (about 5 x 10-11 g): 0.9 x 106 cpm in each case Blood serum. After injection of [125 I]-cytotoxin (Table 2, column 2) the radioactivity underwent a rapid initial decrease followed by a slower decrease. The radioactivity bound to the protein also changed with time. After injection of [1251]-cytotoxin the radioactivity decreased Five min after injection the precipitability ofthe radioactivity corresponded to the value ofthe injected toxin. Nine hr later the value obtained with picric acid was 67'/ and with antibody it was 64% (Table 2, columns 4, 5} This means that in vivo a rather largt: amount of label is removed from the toxin. In mice which were injected with free [1251] the precipitability increased. The increase was 0.9, 0.8, 1.4, 2.2, 3.4 and 9.6% for time intervals of 5 min, 20 min, 1 hr, 3 hr, 6 hr and 24 hr respectively.
765
Pmudomonas aerugbwsa Cytotoxin in Mia
The analysis of the precipitable radioactivity of ["'1]-cytotoxin 9 hr and 24 hr after its injection, using the values of twelve measurements, yields In (y) =1n (0.695) - (0.098 t 0.0097) - t t 0254.
(2)
The correlation coefficient is - 0.954, P < 0.001 ; b = 0.098, resulting in a half-time of7.1 hr. The value of b differs from that of (3) and that of (4) with P < 0.05 . Since a = 0.695, the function accounts for 3.5% ofthe injected radioactivity. The analysis of the non-precipitable radioactivity for 3 hr, 9 hr and 24 hr after injection of [1 Z °I]-cytotoxin, using the values of 18 measurements, results in In (y) =1n (0.608) - (0.160 t 0.017) - t f 0.640.
E
Y Y R )1 E uu
vu O
a 0
E 1 . n u
a
QM .
0 1
3
9
Tims (hours)
a a
24.
FIG. 2 . TIME DEPENDENCE OF RADIOACTIVITY FOR THE PRBCIPITABIE AND NON-PRECIPITABLE FRAC rIONS IN MOUSE SERUM FOLLOWING IN . ADMINISTRATION OF 09 x l()4 cpm [cs'1] COUPLED To CYTOTOXIN OR AS FREE IODEDL
Urcles : precipitable radioactivity following the in', ao of [ 12 'I]-cytotoxin ; squares : nonprocipitable radioactivity following the injection of ' =° I]-cytotoxin ; triangles : non-prccipitable radioactivity following the injection of ["I]. Closed symbols represent geometrical means, open symbols, single determinations. The him enclose the section from which the regression was calculated.
766
F. LUTZ, S . GRIESHABER and I. KÄUFER
The correlation coefficient is 0.912 ; P < 0.001 . The half-time calculated from b is 4.3 hr. The analysis of the non-precipitable radioactivity for 3 hr, 9 hr and 24 hr after injection of ["I], using the values of 18 measurements, results in : In (y) =1n (0.819) - (0.154 t 0.021) - t f 0.813.
(4)
The correlation coefficient is -0.879 ; P < 0.001 . The half-time calculated from b is 4.5 hr. According to the value of a, the function accounts for 4.1% of the injected radioactivity. In Fig. 2 the time dependence of the radioactivity for the precipitable and non-precipitable fractions in mouse serum following the injection of ["'I]-cytotoxin or of free ["-'I] is given for a period of 24 hr. Figure 2 shows that the fraction of non-precipitable radioactivity after injection of ["'I]-cytotoxin is comprised of at least two components, the first of which has a half-time ofseveral min. The fraction of non-precipitable radioactivity after injection offree ["'I] probably consists of two components, the first of which has a half-time of about one min. In both fractions the first component contains 90/ or more ofthe injected radioactivity. Tissue. Five min after injection of the labelled cytotoxin the radioactivity in liver, kidney, spleen and lung was similar to the radioactivity in blood (Table 1} In all other organs it was lower. A continuous decrease, as in blood, was found in most organs during the first hr after injection of [12'I]-cytotoxin. In the abdominal muscle (including peritoneum), uterus and cervical lymph nodes a decrease was measurable after several hr. In the digestive tract, especially in the contents of the stomach, the radioactivity increased during the first hr. After injection of free ["- I] a continuous decrease of radioactivity was observed in all tissues except the intestinal tract (Table 1~ In the upper digestive tract, especially in the contents ofthe stomach, the radioactivity increased rapidly during the first hr. The difference in accumulation of the radioactivity in the intestinal tract for both test groups reveals a relationship between the accumulation in the intestinal tract and the concentration of nonprecipitable radioactivity in the blood. The ratio of the accumulation of radioactivity in the contents of the stomach after injection of ["l I]-cytotoxin versus the accumulation after injection of free ["51] was low when the fraction ofprecipitable radioactivity in serum was high after injection of ["'I]-cytotoxin (Table 2, column 6). Therefore, the accumulation of the radioactivity in the intestinal tract after injection of ["'I]-cytotoxin was caused by free [1251] in the serum. The ratio of radioactivity in serum and blood clot after injection of [' 2 'I]_Cytotoxin was between 1.5 and 1 .9 during the total observation time (Table 2, column 3~ After injection of free [1251] this ratio was between 1 .5 and 1.3. In the brain the radioactivity after injection of either [125 I]-cytotoxin or free [12511 was very low. Excretion. After injection of 2'I-cytotoxin 8T/ of the radioactivity was excreted within 24 hr, 85%in urine and 2% in the faeces (Table 2, column 7~ The excretioncan be described as an exponential function, with a half-time of3.5 hr during the fast hour and a half-time of9 hr in the later period . The radioactivity in urine after injection of [12 '1]-cytotoxin was partially precipitable with picric acid (Table 2, column 8} It was more than 50'/ precipitable in the 20 min and 60 min urine samples. After injection offree [12'1] the precipitability in urine was always smaller than 0.2%. From the precipitability of the radioactivity after injection of [125 1]-cytotoxin, an accumulation of precipitable radioactivity in urine can be calculated over a period of several hr (Table 2, column 10). The highest accumulation was 30-fold in the 20 min urine sample. The cumulative radioactivity which was precipitable in urine during 24 hr after injection of labelled cytotoxin was 2T/ (Table 2, column 9). Analysis by disc electrophoresis showed that the radioactivity co-migrated with cytotoxin (relative mobility 49-92%) and free iodine (relative mobility : 5-48%~
Pseudomonds aeruginosa Cytotoxm in Mice
767
Pathology after i.v. application of lethal amounts of the cytotoxin
Six-nine hr after the injection of 2 jig of the cytotoxin the mice showed an increased hematocrit. Hematocrit values up to 65% were obtained when the animals were in agony. Shortly before death (about one day after cytotoxin injection) an autopsy was performed. Dehydration led to difficulties in removing the skin. The muscle tissues appeared dry. Venous hyperemia could not be detected macroscopically. The stomach was full in all animals. Liver, heart, lung, spleen, kidneys, intestines, bone marrow, gluteus, mesenterial lymphoglandulae and brain were examined histologically . Fatty degeneration, mostly of the central and intermediate regions of the liver lobuli, was frequently seen. In the distal parts of the collecting tubules of the kidney eosinic cylinders with discrete granulocytic reaction and degeneration of epithelial cells were observed. DISCUSSION
The iodinated cytotoxin used in earlier experiments (FRiMMER and SCHARMANN, 1975) suffered from the drawback that more than 50'/ ofits biological activity was lost because the iodination was performed with thechloramine T method. This loss could be eliminated when the lactoperoxidase/glucose oxidase method was used to iodinate the cytotoxin. In distribution studies with enzymatically iodinated cytotoxin it was found that it loses part of its label in vivo. The extent to which this alteration may have an influence on the distribution pattern ofthe labelled cytotoxin in mice can only be conjectured. During 20 min after the injection of [ 125 1]-cytotoxin the protein-bound radioactivity in serum was more than 90'/ . The characteristics of the first component of the protein-bound radioactivity in serum should therefore represent the distribution phase of [ 125 1]_Cytotoxin . The analysis of the radioactivity in serum after injection of [ 125 I]-cytotoxin during the elimination phase reveals a marked difference between protein-bound and non-bound radioactivity. The halftime of the non-bound fraction, 4.3 hr, corresponds to that of the non-bound fraction after injection of free [1251] into control mice (4.5 hr). In contrast, the half-time of the proteinbound radioactivity after injection of [ 125 1]_cytotoxin is 7.1 hr. The parameter of this radioactivity should therefore represent the elimination phase of [ 121 j]-cytotoxin . The data in Table 2 show that in the organs which were examined no specific accumulation was found after i.v. injection of [ 125 I]-cytotoxin. This is in agreement with FRiMMER and SCHARMANN (1975 who observed only a very small uptake of [ 125 1]-cytotoxin by perfused livers from the perfusion medium. In the experiments which are presented here, an accumulation of protein-bound radioactivity was observed during elimination in the urine. This radioactivity co-migrated in electrophoretic analysis according to molecular weight with the cytotoxin. It can be imagined that the cytotoxin could reach toxic concentrations in the kidney during itselimination. In agreement with this are observations on rats by WEINER and REINACHER (1981 After injection of 1 mg cytotoxin per kg body weight, increased concentrations of cytotoxin in the renal tubuli were found, using an immunohistological method. In 1951, OLIVER et al. found that functional disturbances in the kidneys were due to pathological alterations. They showed that toxic acute failure ofthe kidneys in humans was associated with widespread tubular necrosis. After injection of lethal doses ofcytotoxin into mice, we were able to show a degeneration of the epithelial cells in the distal tubuli of the kidney. Similar observations were made by WEINER and REINACI .IER (1981) after Lv. injection of high doses of cytotoxin (1 mg/kg body weight) into rats . Necrosis could be localized in the ascending part of the distal tubuli . Thus, it is probable that the kidney damage is caused by cytotoxin intoxication .
25 (1 .3-92)
093 (0 .86-0.97)
13 (1 .4-1 .7)
.78 0 (0.72-0.87)
1.2 (0.88-1.6)
1 .3 (0.9-20)
1 .8 (1 .4-2.0)
20 (1 .8-2.4)
1.1 (014-2.7)
0.76 (0.57-0.88)
0.95 (0 .44-1.4)
12 (9 .7-23)
4.5 (1 .3-15.0)
5.2 (3 .4-7.1)
1.1 (0 .7-1 .8)
0.49 (0 .27-0.96)
0.77 (0.52-1.4)
0.76 (0.34-1.2)
0.75 (0.41-1.4)
.06 0 (0.03-0.11)
0.71 (0.38-1.3)
.03 0 (0.00-0.12)
Urine
Spleen
Lung
Heart
Uterus
Cervical lymphatic ganglion
Glandulae sublinguales
Stomach
Stomach contents
Ileum
Ileum contents
0.70 (0.63-0.75)
1.5 (12-1.7)
7.2 (3 .1-12.1)
Kidney
.46 0 (0.33-1 .6)
0.80 (0 .64-0.90)
3.0 (21-8.0)
Liver
Abdominal muscle and peritoneum
25 (24-3.0)
[1251]
5.0 (3.7-9.1)
[123j]-toxin
Blood
Location
5 min
0.03 (0.02-0.09) .02 0 (0.00-0.17)
0.17 (0.12-0.24) 0.17 (0.12-017) 0.33 (0.21-0.46)
0.22 (0.06-0.62) 0.10 (0.03-0.19) .11 0 (0.03-0.57)
1.3 (1 .1-1 .9) 0.40 (017-0 .78)
.49 0 (0.43-0.58) .77 0 (0.63-0.89)
6.1 (27-9.9)
0.29 (0.14-0.99)
0.37 (017-0 .37) 0.66 (0.40-0.94)
1.0 (0.67-1.6)
.17 0 (0 .12-0.47)
0.95 (0 .63-1.4) 0.84 (0.65-1.1)
0.59 (0.47-0.69) 0.33 (0.18-0.55)
0.55 (0.26-0.69)
14 .5 (5.7-34)
24 (2.1-3.4)
0.68 (0.20-1.6)
8.4 (3 .4-17)
1.2 (033-24)
1.6 (1.2-2.3)
1.7 (0 .89-2.1)
1.1 (0.59-1.5)
1.3
4.5 (3.4-5.9)
0.96 (0.70-1.6)
1.8 (1 .2-26)
1.3 (0.65-1.9)
1.5 (1 .1-1 .9)
1.1 (1.0-1 .5)
1.3 (0.6-20)
.47 0 (0.32-0.59)
0.28 (015-0 .57)
0.89 (0.75-1.0)
0.76 (0.51-11)
0.67 (0.50-0.85)
.31 0 (017-0.37)
.36 0 (0.31-0.46)
8.7 (24-21)
1.0 (0.61-1.3)
1.3 (0.65-13)
0.60 (0.40-0.67)
.98 0 (0.90-1.1)
0 .54 (0.35-0.71)
0.30 (0 .20-0.38)
.60 0 (0.50-0.73)
0.28 (013-0.34)
0.60 (0.34-0.80)
039 (0.51-0.66)
1.1 (0.9-12)
0.74 (0.57-0.87)
0.90 (0.59-1.0)
24 (1.3-3 .9)
9.9 (4.6-14.6) 1 .8 (1 .0-2.0)
3.8 (3.3-4 .5)
44 (25-62)
22 (29-49) 20 (6.5-49)
40 (15-74)
1.7 (1 .3-25)
0.61 (0.51-0.77)
016 (0 .13-0.47)
0.15 (0.07-0.36)
4.1 (1 .1-14)
1 .5 (0.55-3.6)
.49 0 (0.14-1.6)
0.20 (0.07-0.63)
0.17 (0.06-0.56)
0.15 (0.08-0.40)
14 (10-23)
0.19 (0.07-0.46)
0.61 (0.11-25) 0.04 (0.01-0.26)
1.2 (0 .33-3 .5)
0.20 (0.10-0.38)
0 .08 (0.00-2.25)
0.08 (0 .06-0.16)
.23 0 (0.04-1.0)
0.65 (0 .37-l.3)
0.37 (0.29-0.48)
0.08 (0.02-1.9)
0.03 (0.00-0.29)
0.86 (0.55-1.2)
0.01 (0.00-010)
0.12 (0.06-0.53)
0.10 (0.05-0.14)
0.04 (0.01-011)
027 (0 .05-037)
.08 0 (0 .05-0.17)
0.04 (0.03-0.10)
0.04 (0.02-0.08)
010 (0.17-018)
1.3 (0.55-22)
.02 0 (0.00-0.09)
0.04 (0.01-0.18)
.02 0 (0.00-0.13)
1.8 (1 .3-2.3) .80 0 (0.67-1.02)
.68 0 (0.44-12)
25 (0.03-33)
9.1 (4.4-17)
OA8 (0.29-0.61)
0.11 (0.09-0.16)
0.02 (0.00-0.18)
036 (0.42-0.81)
0.04 (0.03-0.07)
0.07 (0.04-0.13)
tr*
0.01 (0.00-0.03) 0
0.01 (0.00-0.02)
tr*
tr*
tr*
tr'
tr*
tr*
0.03 (0 .01-0.07)
tr*
0.01 (0.00-0.03)
0.01 (0.01-0.02)
24 hr [1251] [1x51]_toxin
0.04 (0.01-0.10)
0.16 (0.12-0.29)
0.12 (0.04-0.28)
3.6 (1 .1-5.6)
7 .7 (26-10.6)
0.37 (0.32-0.49)
0.39 (0.23-0.59)
0.34 (0.17-0.74)
1.1 (0.9-1 .3)
0.34 (0.24-0.74)
[1251]
0.05 (0.01-0.27)
9 hr
[1251]-toxin
1.5 (0.6-8.4)
.81 0 (0.66-1.03)
[1251]
0.59 (0.43-0.69)
1.2 (0.9-1 .6)
3hr 11231]-toxin
20 (0 .7-7 .9)
1.4 (1 .0-1.8)
60 min [1251] [1251]_toxin
FREE [' 2° I] (ABOUT 5 X 10 -11 g) :
19 (1 .6-24)
[1251]
mj cted radioactivity per g body weight
10 -° g) OR
25 (1 .6-3 .7)
["-'I]-toxin
20 min
Radioactivity in organs per g wet weight
TABLE 1 . DISTRIBUTION OF RADIOACf'IVITV IN MICE AFTER 1 .V. APPLICATION OF SUBLETHAL DOSES OF THE [ 125 I]-CVTaroXIN (ABOUT 3 X 0.9 x 106 cpm IN BOTH CASES
.45 0 (0.27-0.76) .03 0 (0 .02-0.12) 1 .0 (0 .33-2Z) .12 0 (0 .Oó-0.21)
.82 0 (0.69-0.99) 020 (0.03-0.73) 0.44 (026-0.74) 0.08 (0.06-0.10)
0.54 (0.50-0.77) 0.02 (0 .00-0.13) 026 (0 .18-0.37) 0.06 (0.03-0.08)
0.71 (0.67-0.75) .61 0 (0.54-0.73) .12 0 (0.02-0Z6) .06 0 (0.04-0.07)
.71 0 (0.68-1 .6) 0.14 (0 .0ó-0Z6) .25 0 (0 .19-0.36) .04 0 (0 .03-0.05)
.42 0 (0 .22-0.56) 0.38 (027-0.66) .17 0 (0 .11-0.37) 0.04
035 (030-0.70) .06 0 (0.01-0.12) .13 0 (0.11-0.15) 0.02 (0 .02-0.03)
.13 0 (0.06-0.50) .27 0 (0.09-0.93) .09 0 (0 .03-0.24) .01 0 (0.01-0.04)
0.33 (0.24--0 .41) 0.21 (0 .09-0.49) .08 0 (0 .03-0.16) .01 0 (0.01-0.03)
.03 0 (0.01-0.21) .28 0 (0.03-3.1) .01 0 (0.00-0.07) tr*
.09 0 (0 .06-0.13) .04 0 (0.02-0.15) .01 0 (0.00-0.07) tr* tr*
0.10 (0.01-3.1) tre
tr*
1.9 t 0.3 1.8 t 04 1.8 t 0.4 1.5 t O.1 1b t 0.1 19 t 0 .2
dot (s)
(6) (6) (6) (6) (6) (6)
rrum IS)
97 t 93 t 83 t 70 t 67 t 82 t
1 1 3 5 13 12
(6) (6) (6) (6) (6) (6)
96 91 82 67 64 72
:11 t 1 t2 t6 t 14 t 10
(6) (6) (6) (6) (6) (6)
Praapitabk with : picric add antitoxin
2.
- OI O S .47 0 1 .9 23 4.1 0.94
: &60-(103 : 5.7 -OOB A3.6 -0 .14 :111 -0L19 .36 AIA -0 : 234 - 040
tid stomach contenta (aller [rïr1taxm appan!ion ) .blood od __ .. . .stomach contenu _ _ (after It- [ 12111 application) blood
2 t 1 (4) 9 t 2 (6) 19 t 7 (8) 44 t 8 (8) 65t8IBIt 85 t 6 16)t
amamt .cmvaed
% or Injected
36 t 10 63 t 8 55±9 30 t 5 18t7 24 t 9
(4) (6) (7) (8) (8) (6)
Predpinbk with picric add
[ 125 1]-BALANCE IN MICE AFTER [ 125 I]-CYTOTOXIN mmCrION
Vahres are means t SD. The number in parentheses is the number of determinations. Based on a plasma volume of 3% body weight (WISH et aL, 1950} t In the faoees were found within 9 hr 0.8 f 0.3% (6) and within 24 hr 1 .8 t O.T/ (5) of the injected radioactivity.
(6) (6) (6) (6) (6) (6)
t 11 t 3 t2 t 10 t 1.1 t0 .1
5 min 20 min 1 hr 3 hr 9hr 24 hr
37 17 11 5 .7 2.4 04
% a(igixted amant*
Tune a0a (njeaion
Blood serum
TABLE
07 . 5 11 18 22 27
: Of~ amamt excreted as predpitabk ndimedvlty
Urine
52 30 13 3 .5 2.0 02
t t t t
3 .8 14 12 1 .8 0.5 .1 t0
(4) (5) (5) (7) (5) (5)
iq)eaad per B body ~t
~Pitabk per B udm
The injxtion volurne was 0.1 mL Values exptas the geometrical toeans of 6-10 mice with [1 '°I]-cytotoxin and of4-5 mice with free [ t =°I]. The range of all values is given in pat+enthews. " Counting rate up to two times background .
Brain
Fat
Caeumcontents
Caecum
770
F. LUTZ, S . GRIESHABER and I. KÄUFER
After injection of ["'I]-labelled exotoxin A from Pseudomonas aeruginosa into mice, the highest concentrations of radioactivity were found in the kidneys (PAVLOVSKIs and SHACKELFORD,1974 ; PAVLovsKIs et al., 1974). Exotoxin A (66,000 daltons) inhibits protein synthesis on the subcellular level . Its LDSO of 0.06-0.081tg/mouse (TAKESHI et al., 1977 ; HommA,1980) is ten times as high as that of the cytotoxin (LUTz,1979). It is of importance for future. experiments to know whether or not the toxicity of the Pseudomonas toxins is
increased if they are injected as a mixture. FRIMMER et al. (1976a) described vascular reactions in rats, especially petechia and exudation, during intoxication shock caused by the cytotoxin, which was purified using an earlier method . The increased hematocrit value of 6-9 hr after the injection of lethal amounts of cytotoxin in mice is an indication that a shock-like reaction occurs in mice when the cytotoxin is injected . However, petechia and exudation were not observed in our experiments. Several factors may be responsible for the different results : (1) the cytotoxin used by FRIMMER et al. (1976a) might have contained impurities which could have damaged vessels, e.g. proteolytic enzyme (Liu,1966) and/or exotoxin A (LIU,1974) ; (2) the doses used by FRIMMER et al. (1976a) were higher than those we used in our experiments. It is likely that we worked with concentrations which were too small to cause vascular damage ; (3) rats and mice may react in a species-specific manner to the cytotoxin intoxication. Mediators of capillary permeability enhancement have been found to differ from one another with respect to effective concentration, reaction mode and effectiveness in different species and tissues (GIERTz,1978~ Experiments investigating the species-dependence of cytotoxin intoxication are in progress . Acknowledgements-This investigation was supported by the Deutsche Forschungsgemeinschaft. We are indebted to Dr . K . O . RXKER for help with the iodinations and Mrs. H. DIEFENBACH-JAGGER for the valuable support in preparing the manuscript . Thanks are due to Prof. Dr. H . RUFEGER for mathematical analysis of our data REFERENCES FRIMMER, M, NEUHOF, H, SCHARMANN, W . and SCHISCHKE, B. (197ÓB) Cardiovascular reactions induced by leucocidin from Pseudomonas aeruginosa . Narnyn-Schmiedeberg's Arch. Pharnweol. 294, 85 . FRIMMER, M, HOMANK J, PErziNGER, E, RUFEGER, U . and ScHARMANN, W . (1976b) Comparative studies on isolated rat hepatocytes and AS-30D hepatoma cells with leucocidin from Pseudomonas aeruginosa. NaunynSchmiedeberg's Arch. Pharmacoi. 295, 63. FRIMMER, M . and SCHARMANN W. (1975) Toxicity of a highly purified leueocidin from Pseudomonas aeruginosa in perfused rat livers. Natnyn-Schmiedeberg's Arch. Phamacol. 288, 123. GiERTz, H . (1978) Mediator substance in shock. In : Verhandhnlgen der Deutschen Gesellschaftfür Pathologic, p.112 (DoHK G, Ed.), Stuttgart : Fischer . HARBOE, N . and INGILD, A. (1973) Immunization, isolation ofitmnunoglobulins, estimation of antibody titre. Scand. J. Immunol. 2, suppl. 1, 161 . HomMA, J. Y. (1980) Roles of exoenzymes and exotoxin in the pathogenicity of Pseudomonas aeruginosa and the development of a new vaccine. Jap . J. exp . Med. 50, 149. KrrzROw, D, BRÜCKLER, J. and BLoBEL, H. (1979) Serologischer Nachweis der "Contagious Equine Metritis" (CEM}B&kterien unter Verwendung protein A-positives Staphylokokken . Tiermztl. Umach. 34, 32. Ltu, P. V. (1966) The roles of various fractions ofPseudomonas aeruginosa in its pathogenesis II. Effects of lecithinase and protease. J. Wwt. Dis . 116, 112. Llu, P. V. (1974) Extracellular toxins of Pseudomonas aeruginosm J. infect . Dis. 130, S94. LowRY, O. H., RosEBRouGK N. J, FARR, A. L. and RANDALL, R. J. (1951) Protein measurement with the folin phenol reagent. J. biol. Chem 193, 265 . LuTz, F. (1979) Purification of a cytotoxic protein from Pseudomonas aeruginosm Toxicon 17, 467. MIYACHI, Y, CHRAMaAcK A, MECKLENBURG, R and LIrsm, M . B . (1973) Preparation and properties of 1 =°I-LHRH. Endocrinology 92, 1725 . OLIVER, J ., MACDowELL, M. and TRAcy, A. (1951) The pathogenesis of acute renal failure associated with traumatic and toxic injury . Renal ischemia, nephrotoxic damage and the ischemuric episode. J . chn . Invest. 30, 1307. PAVLOVSKIS, O. R, CALLAHAN, L. T. III and MEYER, R. D. (1974) Characterization of exotoxin of Pseudomonas aeruginosa. J. infec. Dis. 130, S100.
Pseudomonas aeruginosa Cytotoxin in Mia
771
PAVLOVSKIS, O . R . and SHACKELFORD, A H . (1974) Psedomonas aeruginosa exotoxin in mice : Localization and effect on protein synthesis. IrVect . Immun. 9, 540. SACHS, L . (1978) Angewandte Statistik . 5th Edn, New York, Heidelberg, Berlin : Springer. SCHARMANN, W. (197óa) Formation and isolation of leucocidin from Pseudomonas aeruginosa J. gen. Microbiol. 93, 283. SCHARMANN, W . (1976b) Purification and characterization of leucocidin from Pseudomonas aeruginaw. J . gen . MicTobioL 93, 292. SWANK, R. T. and MUNKRE% K. D . (1971) Molecular weight analysis of oógopeptides by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate. Analyt. Biochem . 39, 462. TAKFSHL, K ., HommA, J. Y, KATO, I. and SArro, H . (1977) Skin necrotizing property of Pseudomonas aeruginosa exotoxin . Jap. J. exp. Med. 47, 323. WEINER, R and REINACHER, M. (1981) Localization of a cytotoxin from Pseudomonas aeruginosa and morphological alterations after its administration in a single lethal dose in rats. Naunyn-Schmiedeberg's Arch. PharmacoL 316, suppl. R46. WISH, L, FURTH, J. and STOREY, R. H . (1950) Direct determinations of plasma, cell, and organ-blood volumes in normal and hypervolemic mice . Proc. Soc. exp . BioL Med . 74, 644 .
0041-MOl/81f6076}-09 m~ O 1981 Perpmm Prey Ltd
CYTOTOXIN FROM PSEUDOMONAS AERUGINOSA IN MICE : DISTRIBUTION RELATED TO PATHOLOGY* F. LUTzt$, S. G~BERt and I. KXUFm§ t Institute of Pharmacology and Toxicology, and §Institute of Veterinary Pathology, Justus-Liebig-University, D-63 Giessen, Federal Republic of Germany (Accepted for publication 12 May 1981)
F. Lurz, SGmPsHAat;Rand 1. KXuFmt. Cytotoxin from Pseudomonaa aengbeosa in mice : distribution related to pathology. Taxiton 19, 763-771,1981 .-The cytotoxin from Pseudomonas aeruginow was labelled with iodine using the lactoperoxidaw/glucose oxidasereaction The relationship between the degree of iodination andcell toxicity wasdetermined . Afteri.v.application of sublethal doses (0 .03leg) of the [I I'I]-cytotoxin (specific activity 311&i/,ug) into mice, 90/of the radioactivity was taken up and distributed among all organs inve ted except the brain. The cytotoxin concentration, calculated on the basis of protein-bound [12 1], washighestin theurine. Lethal i.v. doses(2 pg) of the cytotoxin induced dehydratiomkHistological examination showed degeneration of epithelial cells in the distal kidney tubuli in addition to necrosis of liver lobuli. The damage to kidney tubuli seemsto be responsible for the toxicity of the cytotoxin in truce. INTRODUCTION
A cyTOToxic protein with a molecular weight of25,100 daltons, produced by Pseudomonas aeruginosa (originally isolated from bovine milky is toxic to nearly all mammalian cells (SCHARMANN,.1976b ; FRIMMI?R et A, 1976b ; LUTz,1979~ In studieson isolated cells a dosedependent increase ofcell volume and potassium loss was observed (FRIMME1t et al., 1976b~ However, the mechanism by which the toxin acts is not yet clear. MATERIALS AND METHODS The cytotoxin was prepared from an autolysate of Pseudomonat aerugblosa (LuTz, 1979} Iodination was performed enzymatically according to the method of MtYACHI et al. (19731 with immobilized lactoperoxidase/glucose oxidas (Fazymobead®, Biorad, Mifnchen, FRG} The cytotoxm (50pg) was reacted with unlabelled sodium iodide containing about 0.03 yes (1 kBq) of [1 ='1] (carrier free, Amersham-Buchbr, Braunschweig, FRG) in a total volume of 701d and at molarratios of toxin : iodide from 1 :1 to 1 :10. After various time intervals aliquots of the incubation mixture were tested for precipitable radioactivity using picric acid. The remaining mixture was centrifuged andthesupernatant wastested for toxicity againstbutnan granulocytes in aslide adhesion test (ScHARmANN, 1976x). Seventy per ant of the radioactivity was precipitable within 5 min. The biological activity of the cytotoxin was affected both by the duration of the labelling and the amount of coupled iodine (Fig 1). At one and two moles of iodine per mole of the toxin a loss of its toxicity could not be detected after 5 minof iodination . For thedistribution studies in vivo the cytotoxin wasiodinated with Na[11'I] at aratio of 1 : 1.3 for5 min. To increase theprecipitability to 96-99'/theeluate of the [1:'I]-cytotoxin fraction from a Sephadex G 25 column was dialyzed againstphosphate-buffered aaline. A specific radioactivity of31 f 9pCi/Ng (1 .1 MBq/pg) from nine experiments was calculated. The labelled protein co-migrated with the unlabelled cytotomn in gel electrophoresisand could be stored forfew days at 4°G At thebeginningof each distribution studyprecipitability of the radioactivity of the ["'I]- ytotoxin was controlled. The charge was removed if precipitability was lowerthan 96'/, * A preliminary report has been ptaetlted at the 21st Spring Meeting of the German Pharmacological Society, Mainz 1980 and published in abstract form in Naunyn-Schmiedeberg's Arch. Pharenacol. 1980, 311, suppl, R28. $ Address for corraspondenee. 763
F. LUTZ, S. GRIESHABER and I. KÄUFER
764
Precipitable radioactivity
1001
100
'/.
Relative toxicity against 50 granulocytes
50
0
0 0
5 10 min Iodination time
15
FIG. 1. TIME DEPENDENCE OF THE IODINE LABELLING OF THE CYTOTOXIN AND ITS TOXIC A(TIvrry .
The iodination was performed by the lactoperoxidaw/glucose oxidase reaction. The iodination mixture containeda ratioof toxin : iodide of 1 :1 with traces of [I=sI] . The toxicity ofthecytotoxin for human granulocytes wasdetermined in a slideadhesion test 0---A, radioactivity precipitable with all picric acid ; 0-", 50'/ of the granulocytes were swollen within 30min ; 0-0, granulocytes were swollen within 30 min.
Protein binding of radioactivity was determined by precipitation with equal amounts of saturated picric acid in the presence of bovine serum albumin. In the case of serum, the reaction of toxin with rabbit antitoxin was also utilized . Tests were performed with antitoxin in solution or coupled to protein A-positive Staphylococci (KrrzRow et al., 1979). If the antitoxin in solution was used, non-labelled cytotoxin was added to the incubation vessel at the end of the 1 hr incubation to increase the total sediment After washing the sediment the radioactivity was determined. A Packard Autogammata counter (efficiency 68%) was used to measure the radioactivity. Protein was determined by the method of LOWRY et al. (1951 Sodium dodecyl sulphate polyacrylamide gel electrophoresis was performed as described by SWANK and MUNKRES (1971 Antitoxin was prepared as described by HARBOE and INOILD (1973). Female HAN-NMRI mice of about30 gbody weight,bred in ourfacilities,were used . Fordistribution studies, the drinking water contained 0.42'/ NaCl and 0.05% NaI and wasprovided ad fiNtwn for two weeks. Urine was collected by means of polyethylene-ooated filter paper on the cage floor. The mice were anesthetized with ether before removal of the organs . The radioactivity data of serum were analyzed by the method of least squares, using the linear function :
(1) In (y) = In (a) + (b t sr) - t t %( y).., where y is the quotient of cpm found per g serum and cpm injected per g body weight The terms in (a) and b are parameters of the function, t is the time after injection of radioactivity, sb is the standard deviation of b and 3Wy).x Is the standard deviation of the function . The half-time is given by 1/b - In(2} The remaining statistical analysis was carried out using the method of SACHS (1978). All standard deviations except sr are standard deviations of single values. RESULTS
Distribution of radioactivity after application of small amounts of [ 125 1]-eytotoxin (about 3 x 10 a g) or free [1231] (about 5 x 10-11 g): 0.9 x 106 cpm in each case Blood serum. After injection of [125 I]-cytotoxin (Table 2, column 2) the radioactivity underwent a rapid initial decrease followed by a slower decrease. The radioactivity bound to the protein also changed with time. After injection of [1251]-cytotoxin the radioactivity decreased Five min after injection the precipitability ofthe radioactivity corresponded to the value ofthe injected toxin. Nine hr later the value obtained with picric acid was 67'/ and with antibody it was 64% (Table 2, columns 4, 5} This means that in vivo a rather largt: amount of label is removed from the toxin. In mice which were injected with free [1251] the precipitability increased. The increase was 0.9, 0.8, 1.4, 2.2, 3.4 and 9.6% for time intervals of 5 min, 20 min, 1 hr, 3 hr, 6 hr and 24 hr respectively.
765
Pmudomonas aerugbwsa Cytotoxin in Mia
The analysis of the precipitable radioactivity of ["'1]-cytotoxin 9 hr and 24 hr after its injection, using the values of twelve measurements, yields In (y) =1n (0.695) - (0.098 t 0.0097) - t t 0254.
(2)
The correlation coefficient is - 0.954, P < 0.001 ; b = 0.098, resulting in a half-time of7.1 hr. The value of b differs from that of (3) and that of (4) with P < 0.05 . Since a = 0.695, the function accounts for 3.5% ofthe injected radioactivity. The analysis of the non-precipitable radioactivity for 3 hr, 9 hr and 24 hr after injection of [1 Z °I]-cytotoxin, using the values of 18 measurements, results in In (y) =1n (0.608) - (0.160 t 0.017) - t f 0.640.
E
Y Y R )1 E uu
vu O
a 0
E 1 . n u
a
QM .
0 1
3
9
Tims (hours)
a a
24.
FIG. 2 . TIME DEPENDENCE OF RADIOACTIVITY FOR THE PRBCIPITABIE AND NON-PRECIPITABLE FRAC rIONS IN MOUSE SERUM FOLLOWING IN . ADMINISTRATION OF 09 x l()4 cpm [cs'1] COUPLED To CYTOTOXIN OR AS FREE IODEDL
Urcles : precipitable radioactivity following the in', ao of [ 12 'I]-cytotoxin ; squares : nonprocipitable radioactivity following the injection of ' =° I]-cytotoxin ; triangles : non-prccipitable radioactivity following the injection of ["I]. Closed symbols represent geometrical means, open symbols, single determinations. The him enclose the section from which the regression was calculated.
766
F. LUTZ, S . GRIESHABER and I. KÄUFER
The correlation coefficient is 0.912 ; P < 0.001 . The half-time calculated from b is 4.3 hr. The analysis of the non-precipitable radioactivity for 3 hr, 9 hr and 24 hr after injection of ["I], using the values of 18 measurements, results in : In (y) =1n (0.819) - (0.154 t 0.021) - t f 0.813.
(4)
The correlation coefficient is -0.879 ; P < 0.001 . The half-time calculated from b is 4.5 hr. According to the value of a, the function accounts for 4.1% of the injected radioactivity. In Fig. 2 the time dependence of the radioactivity for the precipitable and non-precipitable fractions in mouse serum following the injection of ["'I]-cytotoxin or of free ["-'I] is given for a period of 24 hr. Figure 2 shows that the fraction of non-precipitable radioactivity after injection of ["'I]-cytotoxin is comprised of at least two components, the first of which has a half-time ofseveral min. The fraction of non-precipitable radioactivity after injection offree ["'I] probably consists of two components, the first of which has a half-time of about one min. In both fractions the first component contains 90/ or more ofthe injected radioactivity. Tissue. Five min after injection of the labelled cytotoxin the radioactivity in liver, kidney, spleen and lung was similar to the radioactivity in blood (Table 1} In all other organs it was lower. A continuous decrease, as in blood, was found in most organs during the first hr after injection of [12'I]-cytotoxin. In the abdominal muscle (including peritoneum), uterus and cervical lymph nodes a decrease was measurable after several hr. In the digestive tract, especially in the contents of the stomach, the radioactivity increased during the first hr. After injection of free ["- I] a continuous decrease of radioactivity was observed in all tissues except the intestinal tract (Table 1~ In the upper digestive tract, especially in the contents ofthe stomach, the radioactivity increased rapidly during the first hr. The difference in accumulation of the radioactivity in the intestinal tract for both test groups reveals a relationship between the accumulation in the intestinal tract and the concentration of nonprecipitable radioactivity in the blood. The ratio of the accumulation of radioactivity in the contents of the stomach after injection of ["l I]-cytotoxin versus the accumulation after injection of free ["51] was low when the fraction ofprecipitable radioactivity in serum was high after injection of ["'I]-cytotoxin (Table 2, column 6). Therefore, the accumulation of the radioactivity in the intestinal tract after injection of ["'I]-cytotoxin was caused by free [1251] in the serum. The ratio of radioactivity in serum and blood clot after injection of [' 2 'I]_Cytotoxin was between 1.5 and 1 .9 during the total observation time (Table 2, column 3~ After injection of free [1251] this ratio was between 1 .5 and 1.3. In the brain the radioactivity after injection of either [125 I]-cytotoxin or free [12511 was very low. Excretion. After injection of 2'I-cytotoxin 8T/ of the radioactivity was excreted within 24 hr, 85%in urine and 2% in the faeces (Table 2, column 7~ The excretioncan be described as an exponential function, with a half-time of3.5 hr during the fast hour and a half-time of9 hr in the later period . The radioactivity in urine after injection of [12 '1]-cytotoxin was partially precipitable with picric acid (Table 2, column 8} It was more than 50'/ precipitable in the 20 min and 60 min urine samples. After injection offree [12'1] the precipitability in urine was always smaller than 0.2%. From the precipitability of the radioactivity after injection of [125 1]-cytotoxin, an accumulation of precipitable radioactivity in urine can be calculated over a period of several hr (Table 2, column 10). The highest accumulation was 30-fold in the 20 min urine sample. The cumulative radioactivity which was precipitable in urine during 24 hr after injection of labelled cytotoxin was 2T/ (Table 2, column 9). Analysis by disc electrophoresis showed that the radioactivity co-migrated with cytotoxin (relative mobility 49-92%) and free iodine (relative mobility : 5-48%~
Pseudomonds aeruginosa Cytotoxm in Mice
767
Pathology after i.v. application of lethal amounts of the cytotoxin
Six-nine hr after the injection of 2 jig of the cytotoxin the mice showed an increased hematocrit. Hematocrit values up to 65% were obtained when the animals were in agony. Shortly before death (about one day after cytotoxin injection) an autopsy was performed. Dehydration led to difficulties in removing the skin. The muscle tissues appeared dry. Venous hyperemia could not be detected macroscopically. The stomach was full in all animals. Liver, heart, lung, spleen, kidneys, intestines, bone marrow, gluteus, mesenterial lymphoglandulae and brain were examined histologically . Fatty degeneration, mostly of the central and intermediate regions of the liver lobuli, was frequently seen. In the distal parts of the collecting tubules of the kidney eosinic cylinders with discrete granulocytic reaction and degeneration of epithelial cells were observed. DISCUSSION
The iodinated cytotoxin used in earlier experiments (FRiMMER and SCHARMANN, 1975) suffered from the drawback that more than 50'/ ofits biological activity was lost because the iodination was performed with thechloramine T method. This loss could be eliminated when the lactoperoxidase/glucose oxidase method was used to iodinate the cytotoxin. In distribution studies with enzymatically iodinated cytotoxin it was found that it loses part of its label in vivo. The extent to which this alteration may have an influence on the distribution pattern ofthe labelled cytotoxin in mice can only be conjectured. During 20 min after the injection of [ 125 1]-cytotoxin the protein-bound radioactivity in serum was more than 90'/ . The characteristics of the first component of the protein-bound radioactivity in serum should therefore represent the distribution phase of [ 125 1]_Cytotoxin . The analysis of the radioactivity in serum after injection of [ 125 I]-cytotoxin during the elimination phase reveals a marked difference between protein-bound and non-bound radioactivity. The halftime of the non-bound fraction, 4.3 hr, corresponds to that of the non-bound fraction after injection of free [1251] into control mice (4.5 hr). In contrast, the half-time of the proteinbound radioactivity after injection of [ 125 1]_cytotoxin is 7.1 hr. The parameter of this radioactivity should therefore represent the elimination phase of [ 121 j]-cytotoxin . The data in Table 2 show that in the organs which were examined no specific accumulation was found after i.v. injection of [ 125 I]-cytotoxin. This is in agreement with FRiMMER and SCHARMANN (1975 who observed only a very small uptake of [ 125 1]-cytotoxin by perfused livers from the perfusion medium. In the experiments which are presented here, an accumulation of protein-bound radioactivity was observed during elimination in the urine. This radioactivity co-migrated in electrophoretic analysis according to molecular weight with the cytotoxin. It can be imagined that the cytotoxin could reach toxic concentrations in the kidney during itselimination. In agreement with this are observations on rats by WEINER and REINACHER (1981 After injection of 1 mg cytotoxin per kg body weight, increased concentrations of cytotoxin in the renal tubuli were found, using an immunohistological method. In 1951, OLIVER et al. found that functional disturbances in the kidneys were due to pathological alterations. They showed that toxic acute failure ofthe kidneys in humans was associated with widespread tubular necrosis. After injection of lethal doses ofcytotoxin into mice, we were able to show a degeneration of the epithelial cells in the distal tubuli of the kidney. Similar observations were made by WEINER and REINACI .IER (1981) after Lv. injection of high doses of cytotoxin (1 mg/kg body weight) into rats . Necrosis could be localized in the ascending part of the distal tubuli . Thus, it is probable that the kidney damage is caused by cytotoxin intoxication .
25 (1 .3-92)
093 (0 .86-0.97)
13 (1 .4-1 .7)
.78 0 (0.72-0.87)
1.2 (0.88-1.6)
1 .3 (0.9-20)
1 .8 (1 .4-2.0)
20 (1 .8-2.4)
1.1 (014-2.7)
0.76 (0.57-0.88)
0.95 (0 .44-1.4)
12 (9 .7-23)
4.5 (1 .3-15.0)
5.2 (3 .4-7.1)
1.1 (0 .7-1 .8)
0.49 (0 .27-0.96)
0.77 (0.52-1.4)
0.76 (0.34-1.2)
0.75 (0.41-1.4)
.06 0 (0.03-0.11)
0.71 (0.38-1.3)
.03 0 (0.00-0.12)
Urine
Spleen
Lung
Heart
Uterus
Cervical lymphatic ganglion
Glandulae sublinguales
Stomach
Stomach contents
Ileum
Ileum contents
0.70 (0.63-0.75)
1.5 (12-1.7)
7.2 (3 .1-12.1)
Kidney
.46 0 (0.33-1 .6)
0.80 (0 .64-0.90)
3.0 (21-8.0)
Liver
Abdominal muscle and peritoneum
25 (24-3.0)
[1251]
5.0 (3.7-9.1)
[123j]-toxin
Blood
Location
5 min
0.03 (0.02-0.09) .02 0 (0.00-0.17)
0.17 (0.12-0.24) 0.17 (0.12-017) 0.33 (0.21-0.46)
0.22 (0.06-0.62) 0.10 (0.03-0.19) .11 0 (0.03-0.57)
1.3 (1 .1-1 .9) 0.40 (017-0 .78)
.49 0 (0.43-0.58) .77 0 (0.63-0.89)
6.1 (27-9.9)
0.29 (0.14-0.99)
0.37 (017-0 .37) 0.66 (0.40-0.94)
1.0 (0.67-1.6)
.17 0 (0 .12-0.47)
0.95 (0 .63-1.4) 0.84 (0.65-1.1)
0.59 (0.47-0.69) 0.33 (0.18-0.55)
0.55 (0.26-0.69)
14 .5 (5.7-34)
24 (2.1-3.4)
0.68 (0.20-1.6)
8.4 (3 .4-17)
1.2 (033-24)
1.6 (1.2-2.3)
1.7 (0 .89-2.1)
1.1 (0.59-1.5)
1.3
4.5 (3.4-5.9)
0.96 (0.70-1.6)
1.8 (1 .2-26)
1.3 (0.65-1.9)
1.5 (1 .1-1 .9)
1.1 (1.0-1 .5)
1.3 (0.6-20)
.47 0 (0.32-0.59)
0.28 (015-0 .57)
0.89 (0.75-1.0)
0.76 (0.51-11)
0.67 (0.50-0.85)
.31 0 (017-0.37)
.36 0 (0.31-0.46)
8.7 (24-21)
1.0 (0.61-1.3)
1.3 (0.65-13)
0.60 (0.40-0.67)
.98 0 (0.90-1.1)
0 .54 (0.35-0.71)
0.30 (0 .20-0.38)
.60 0 (0.50-0.73)
0.28 (013-0.34)
0.60 (0.34-0.80)
039 (0.51-0.66)
1.1 (0.9-12)
0.74 (0.57-0.87)
0.90 (0.59-1.0)
24 (1.3-3 .9)
9.9 (4.6-14.6) 1 .8 (1 .0-2.0)
3.8 (3.3-4 .5)
44 (25-62)
22 (29-49) 20 (6.5-49)
40 (15-74)
1.7 (1 .3-25)
0.61 (0.51-0.77)
016 (0 .13-0.47)
0.15 (0.07-0.36)
4.1 (1 .1-14)
1 .5 (0.55-3.6)
.49 0 (0.14-1.6)
0.20 (0.07-0.63)
0.17 (0.06-0.56)
0.15 (0.08-0.40)
14 (10-23)
0.19 (0.07-0.46)
0.61 (0.11-25) 0.04 (0.01-0.26)
1.2 (0 .33-3 .5)
0.20 (0.10-0.38)
0 .08 (0.00-2.25)
0.08 (0 .06-0.16)
.23 0 (0.04-1.0)
0.65 (0 .37-l.3)
0.37 (0.29-0.48)
0.08 (0.02-1.9)
0.03 (0.00-0.29)
0.86 (0.55-1.2)
0.01 (0.00-010)
0.12 (0.06-0.53)
0.10 (0.05-0.14)
0.04 (0.01-011)
027 (0 .05-037)
.08 0 (0 .05-0.17)
0.04 (0.03-0.10)
0.04 (0.02-0.08)
010 (0.17-018)
1.3 (0.55-22)
.02 0 (0.00-0.09)
0.04 (0.01-0.18)
.02 0 (0.00-0.13)
1.8 (1 .3-2.3) .80 0 (0.67-1.02)
.68 0 (0.44-12)
25 (0.03-33)
9.1 (4.4-17)
OA8 (0.29-0.61)
0.11 (0.09-0.16)
0.02 (0.00-0.18)
036 (0.42-0.81)
0.04 (0.03-0.07)
0.07 (0.04-0.13)
tr*
0.01 (0.00-0.03) 0
0.01 (0.00-0.02)
tr*
tr*
tr*
tr'
tr*
tr*
0.03 (0 .01-0.07)
tr*
0.01 (0.00-0.03)
0.01 (0.01-0.02)
24 hr [1251] [1x51]_toxin
0.04 (0.01-0.10)
0.16 (0.12-0.29)
0.12 (0.04-0.28)
3.6 (1 .1-5.6)
7 .7 (26-10.6)
0.37 (0.32-0.49)
0.39 (0.23-0.59)
0.34 (0.17-0.74)
1.1 (0.9-1 .3)
0.34 (0.24-0.74)
[1251]
0.05 (0.01-0.27)
9 hr
[1251]-toxin
1.5 (0.6-8.4)
.81 0 (0.66-1.03)
[1251]
0.59 (0.43-0.69)
1.2 (0.9-1 .6)
3hr 11231]-toxin
20 (0 .7-7 .9)
1.4 (1 .0-1.8)
60 min [1251] [1251]_toxin
FREE [' 2° I] (ABOUT 5 X 10 -11 g) :
19 (1 .6-24)
[1251]
mj cted radioactivity per g body weight
10 -° g) OR
25 (1 .6-3 .7)
["-'I]-toxin
20 min
Radioactivity in organs per g wet weight
TABLE 1 . DISTRIBUTION OF RADIOACf'IVITV IN MICE AFTER 1 .V. APPLICATION OF SUBLETHAL DOSES OF THE [ 125 I]-CVTaroXIN (ABOUT 3 X 0.9 x 106 cpm IN BOTH CASES
.45 0 (0.27-0.76) .03 0 (0 .02-0.12) 1 .0 (0 .33-2Z) .12 0 (0 .Oó-0.21)
.82 0 (0.69-0.99) 020 (0.03-0.73) 0.44 (026-0.74) 0.08 (0.06-0.10)
0.54 (0.50-0.77) 0.02 (0 .00-0.13) 026 (0 .18-0.37) 0.06 (0.03-0.08)
0.71 (0.67-0.75) .61 0 (0.54-0.73) .12 0 (0.02-0Z6) .06 0 (0.04-0.07)
.71 0 (0.68-1 .6) 0.14 (0 .0ó-0Z6) .25 0 (0 .19-0.36) .04 0 (0 .03-0.05)
.42 0 (0 .22-0.56) 0.38 (027-0.66) .17 0 (0 .11-0.37) 0.04
035 (030-0.70) .06 0 (0.01-0.12) .13 0 (0.11-0.15) 0.02 (0 .02-0.03)
.13 0 (0.06-0.50) .27 0 (0.09-0.93) .09 0 (0 .03-0.24) .01 0 (0.01-0.04)
0.33 (0.24--0 .41) 0.21 (0 .09-0.49) .08 0 (0 .03-0.16) .01 0 (0.01-0.03)
.03 0 (0.01-0.21) .28 0 (0.03-3.1) .01 0 (0.00-0.07) tr*
.09 0 (0 .06-0.13) .04 0 (0.02-0.15) .01 0 (0.00-0.07) tr* tr*
0.10 (0.01-3.1) tre
tr*
1.9 t 0.3 1.8 t 04 1.8 t 0.4 1.5 t O.1 1b t 0.1 19 t 0 .2
dot (s)
(6) (6) (6) (6) (6) (6)
rrum IS)
97 t 93 t 83 t 70 t 67 t 82 t
1 1 3 5 13 12
(6) (6) (6) (6) (6) (6)
96 91 82 67 64 72
:11 t 1 t2 t6 t 14 t 10
(6) (6) (6) (6) (6) (6)
Praapitabk with : picric add antitoxin
2.
- OI O S .47 0 1 .9 23 4.1 0.94
: &60-(103 : 5.7 -OOB A3.6 -0 .14 :111 -0L19 .36 AIA -0 : 234 - 040
tid stomach contenta (aller [rïr1taxm appan!ion ) .blood od __ .. . .stomach contenu _ _ (after It- [ 12111 application) blood
2 t 1 (4) 9 t 2 (6) 19 t 7 (8) 44 t 8 (8) 65t8IBIt 85 t 6 16)t
amamt .cmvaed
% or Injected
36 t 10 63 t 8 55±9 30 t 5 18t7 24 t 9
(4) (6) (7) (8) (8) (6)
Predpinbk with picric add
[ 125 1]-BALANCE IN MICE AFTER [ 125 I]-CYTOTOXIN mmCrION
Vahres are means t SD. The number in parentheses is the number of determinations. Based on a plasma volume of 3% body weight (WISH et aL, 1950} t In the faoees were found within 9 hr 0.8 f 0.3% (6) and within 24 hr 1 .8 t O.T/ (5) of the injected radioactivity.
(6) (6) (6) (6) (6) (6)
t 11 t 3 t2 t 10 t 1.1 t0 .1
5 min 20 min 1 hr 3 hr 9hr 24 hr
37 17 11 5 .7 2.4 04
% a(igixted amant*
Tune a0a (njeaion
Blood serum
TABLE
07 . 5 11 18 22 27
: Of~ amamt excreted as predpitabk ndimedvlty
Urine
52 30 13 3 .5 2.0 02
t t t t
3 .8 14 12 1 .8 0.5 .1 t0
(4) (5) (5) (7) (5) (5)
iq)eaad per B body ~t
~Pitabk per B udm
The injxtion volurne was 0.1 mL Values exptas the geometrical toeans of 6-10 mice with [1 '°I]-cytotoxin and of4-5 mice with free [ t =°I]. The range of all values is given in pat+enthews. " Counting rate up to two times background .
Brain
Fat
Caeumcontents
Caecum
770
F. LUTZ, S . GRIESHABER and I. KÄUFER
After injection of ["'I]-labelled exotoxin A from Pseudomonas aeruginosa into mice, the highest concentrations of radioactivity were found in the kidneys (PAVLOVSKIs and SHACKELFORD,1974 ; PAVLovsKIs et al., 1974). Exotoxin A (66,000 daltons) inhibits protein synthesis on the subcellular level . Its LDSO of 0.06-0.081tg/mouse (TAKESHI et al., 1977 ; HommA,1980) is ten times as high as that of the cytotoxin (LUTz,1979). It is of importance for future. experiments to know whether or not the toxicity of the Pseudomonas toxins is
increased if they are injected as a mixture. FRIMMER et al. (1976a) described vascular reactions in rats, especially petechia and exudation, during intoxication shock caused by the cytotoxin, which was purified using an earlier method . The increased hematocrit value of 6-9 hr after the injection of lethal amounts of cytotoxin in mice is an indication that a shock-like reaction occurs in mice when the cytotoxin is injected . However, petechia and exudation were not observed in our experiments. Several factors may be responsible for the different results : (1) the cytotoxin used by FRIMMER et al. (1976a) might have contained impurities which could have damaged vessels, e.g. proteolytic enzyme (Liu,1966) and/or exotoxin A (LIU,1974) ; (2) the doses used by FRIMMER et al. (1976a) were higher than those we used in our experiments. It is likely that we worked with concentrations which were too small to cause vascular damage ; (3) rats and mice may react in a species-specific manner to the cytotoxin intoxication. Mediators of capillary permeability enhancement have been found to differ from one another with respect to effective concentration, reaction mode and effectiveness in different species and tissues (GIERTz,1978~ Experiments investigating the species-dependence of cytotoxin intoxication are in progress . Acknowledgements-This investigation was supported by the Deutsche Forschungsgemeinschaft. We are indebted to Dr . K . O . RXKER for help with the iodinations and Mrs. H. DIEFENBACH-JAGGER for the valuable support in preparing the manuscript . Thanks are due to Prof. Dr. H . RUFEGER for mathematical analysis of our data REFERENCES FRIMMER, M, NEUHOF, H, SCHARMANN, W . and SCHISCHKE, B. (197ÓB) Cardiovascular reactions induced by leucocidin from Pseudomonas aeruginosa . Narnyn-Schmiedeberg's Arch. Pharnweol. 294, 85 . FRIMMER, M, HOMANK J, PErziNGER, E, RUFEGER, U . and ScHARMANN, W . (1976b) Comparative studies on isolated rat hepatocytes and AS-30D hepatoma cells with leucocidin from Pseudomonas aeruginosa. NaunynSchmiedeberg's Arch. Pharmacoi. 295, 63. FRIMMER, M . and SCHARMANN W. (1975) Toxicity of a highly purified leueocidin from Pseudomonas aeruginosa in perfused rat livers. Natnyn-Schmiedeberg's Arch. Phamacol. 288, 123. GiERTz, H . (1978) Mediator substance in shock. In : Verhandhnlgen der Deutschen Gesellschaftfür Pathologic, p.112 (DoHK G, Ed.), Stuttgart : Fischer . HARBOE, N . and INGILD, A. (1973) Immunization, isolation ofitmnunoglobulins, estimation of antibody titre. Scand. J. Immunol. 2, suppl. 1, 161 . HomMA, J. Y. (1980) Roles of exoenzymes and exotoxin in the pathogenicity of Pseudomonas aeruginosa and the development of a new vaccine. Jap . J. exp . Med. 50, 149. KrrzROw, D, BRÜCKLER, J. and BLoBEL, H. (1979) Serologischer Nachweis der "Contagious Equine Metritis" (CEM}B&kterien unter Verwendung protein A-positives Staphylokokken . Tiermztl. Umach. 34, 32. Ltu, P. V. (1966) The roles of various fractions ofPseudomonas aeruginosa in its pathogenesis II. Effects of lecithinase and protease. J. Wwt. Dis . 116, 112. Llu, P. V. (1974) Extracellular toxins of Pseudomonas aeruginosm J. infect . Dis. 130, S94. LowRY, O. H., RosEBRouGK N. J, FARR, A. L. and RANDALL, R. J. (1951) Protein measurement with the folin phenol reagent. J. biol. Chem 193, 265 . LuTz, F. (1979) Purification of a cytotoxic protein from Pseudomonas aeruginosm Toxicon 17, 467. MIYACHI, Y, CHRAMaAcK A, MECKLENBURG, R and LIrsm, M . B . (1973) Preparation and properties of 1 =°I-LHRH. Endocrinology 92, 1725 . OLIVER, J ., MACDowELL, M. and TRAcy, A. (1951) The pathogenesis of acute renal failure associated with traumatic and toxic injury . Renal ischemia, nephrotoxic damage and the ischemuric episode. J . chn . Invest. 30, 1307. PAVLOVSKIS, O. R, CALLAHAN, L. T. III and MEYER, R. D. (1974) Characterization of exotoxin of Pseudomonas aeruginosa. J. infec. Dis. 130, S100.
Pseudomonas aeruginosa Cytotoxin in Mia
771
PAVLOVSKIS, O . R . and SHACKELFORD, A H . (1974) Psedomonas aeruginosa exotoxin in mice : Localization and effect on protein synthesis. IrVect . Immun. 9, 540. SACHS, L . (1978) Angewandte Statistik . 5th Edn, New York, Heidelberg, Berlin : Springer. SCHARMANN, W. (197óa) Formation and isolation of leucocidin from Pseudomonas aeruginosa J. gen. Microbiol. 93, 283. SCHARMANN, W . (1976b) Purification and characterization of leucocidin from Pseudomonas aeruginaw. J . gen . MicTobioL 93, 292. SWANK, R. T. and MUNKRE% K. D . (1971) Molecular weight analysis of oógopeptides by electrophoresis in polyacrylamide gel with sodium dodecyl sulfate. Analyt. Biochem . 39, 462. TAKFSHL, K ., HommA, J. Y, KATO, I. and SArro, H . (1977) Skin necrotizing property of Pseudomonas aeruginosa exotoxin . Jap. J. exp. Med. 47, 323. WEINER, R and REINACHER, M. (1981) Localization of a cytotoxin from Pseudomonas aeruginosa and morphological alterations after its administration in a single lethal dose in rats. Naunyn-Schmiedeberg's Arch. PharmacoL 316, suppl. R46. WISH, L, FURTH, J. and STOREY, R. H . (1950) Direct determinations of plasma, cell, and organ-blood volumes in normal and hypervolemic mice . Proc. Soc. exp . BioL Med . 74, 644 .