Pharmacological Research, Vol. 26, No . 3, 1992
27 7
PRESENCE OF TOBRAMYCIN IN KIDNEYS AND PLACENTAE OF SPRAGUE-DAWLEY RAT FETUSES AND NEWBORNS FOLLOWING IN UTERO EXPOSURE A . M . FERRINI*, P . AURELI*, C . RICCIARDIt, A . V . STAZIt, C . MACRIt and A . MANTOVANIt *Laboratorio Alimenti and tLaboratorio di Tossicologia Comparata ed Ecotossicologia, Istituto Superiore di Sanita, viale Regina Elena, 299, 00161 Roma, Italy Received in final form I April 1992
SUMMARY The maternal-fetal transfer of tobramycin (TBM) was investigated in the rat by means of a microbiological assay, to assess the presence and amount in the kidneys and placentae of fetuses at gestational day (GD) 20, in the kidneys of newborns 6 and 11 days after the end of treatment and in the kidneys of the dams . In the qualitative assay, pregnant rats were injected i .p . with 0, 30, 60 mg/kg b .w . of TBM on GD 10-19 . A group of dams treated in parallel with 30 mg/kg b .w . was utilized for a microbiological semiquantitative assay . All litters contained some fetuses showing no detectable TBM accumulation in either kidney or in placentae : at 30 mg/kg/b .w . accumulation appeared more prevalent in placenta than in the kidneys of the corresponding fetuses, as confirmed also by the semiquantitative assay . Some newborns (about 6%) of both groups showed detectable renal TBM residues on the 6th and on the 11th day after the end of treatment . The frequency of newborns showing residues was not obviously related to the dose or the day of sampling, and the concentrations of TBM found were comparable to those observed in fetuses . It is possible that they represent a particularly sensitive subgroup . KEY WORDS :
offspring, tobramycin, kidney, placental transfer .
INTRODUCTION The kidney can be a target organ for developmental toxicants, as shown by studies concerning environmental agents [1] or aminoglycoside gentamycin [2,3] . The nephrotoxic aminoglycoside tobramycin (TBM) is apparently devoid of readily detectable adverse developmental effects [4] although it has been demonstrated to be present in the kidneys and placentae of human fetuses from treated mothers [5] . Moreover, TBM elicits microscopical alterations of renal development in SpragueDawley rat fetuses on gestational day (GD) 20 and in newborns on postnatal day 1043-6618/92/070277-08/$08 .00/0
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(PD) 9, exposed in utero to 60 or 30 mg/kg from GD 10 to GD 19, i .e . the period of major renal organogenesis [6] . The experiments described here were performed to assess the renal and placental accumulation of TBM in rat conceptuses, as well as the persistence of TBM in early post-natal life, following a treatment schedule that was identical to the one leading to micromorphological alterations . The presence and amount of TBM were respectively defined by qualitative and semiquantitative microbiological methods [see, e .g ., 7] to detect biologically active residues . The preliminary results of our study were presented at the 18th European Teratology Society Conference [8] .
MATERIALS AND METHODS Animals
Nulliparous Sprague-Dawley female rats weighing 180-200 g were purchased from Charles River (Calco, Como, Italy) and held for one week for acclimatization . They were kept in a self-contained unit under the following environmental conditions : 22 ± 0 .5 ° C ; humidity 55% ; 12-14 air changes/h ; 12 :12 light/dark photoperiod . Drinking water and food (Open Formula, D .ri Piccioni, Brescia, Italy) were available ad libitum . Compound
Tobramycin (QA179J, batch Y4855, activity : 946 mg/g) was obtained from Eli Lilly Italia S .p .A . (Sesto Fiorentino, Italy) . Doses
Doses were : 30 mg/kg b .w . (group A) ; 60 mg/kg b .w . (group B) ; 30 mg/kg b .w . (group S) of TBM vehicled in a sterile saline solution (I ml solution/300 g b .w .) ; control group (group C), only vehicle solution . Treatment Schedule
The females were mated (2 :1) to adult males and the day of a sperm positive vaginal smear was defined as GD 0 . On GD 10 they were weighed and assigned to the experimental groups . From GD 10 to GD 19 (around 09 .30 a .m . each day) they were treated i .p . to allow a rapid absorption of the drug . On GD 20 (i .e . the day after the last treatment) some of the females in each group were overanaesthetized by i .p . injection of sodium pentothal followed by cervical dislocation . The maternal kidneys, fetal kidneys and placentae were collected in sterile conditions (1st sampling) . Each fetus was sexed, weighed, identified and overanesthetized before sampling . The other dams were allowed to deliver the litters (most of the rats gave birth to their litters on GD 22) . On the 6th and on the 11th day following the last treatment (2nd and 3rd sampling), the dams and their newborns were overanesthetized and killed by cervical dislocation ; the maternal and neonatal kidneys were then collected in sterile conditions . Each newborn was sexed, weighed and identified . The parallel group (group S) received 30 mg/kg b .w ., by the same treatment
Pharmacological Research, Vol. 26, No . 3, 1992
27 9
schedule as above, and the samples were utilized to perform the semiquantitative analysis of TBM residues . For an overview of the experimental design see Table 1 . Since they had been extensively studied in a previous experiment [6] no parameters of maternal or developmental toxicity were monitored in the present study, except cage-side and external observations . All samples were immediately kept at 4°C and assayed within 2 h . Microbiological method
Antibiotic activity was determined by the agar diffusion method with Bacillus subtilis ATCC 6633 (Strain BGA) as the reference strain : this strain is also used in the German Hemmstofftest, because of its sensitivity [9] . Standard curve
The linear concentration range of standard dilutions of TBM was determined according to the standard curve procedure as described by Cooper [10] . An accurately determined amount of TBM was dissolved to give a stock solution containing 1000 ug/ml . From this stock solution, 10 concentrations ranging from 200 to 0 .25 ug/ml were obtained by successive dilutions . Paper disks (13 mm diameter) were soaked with 100 ml of each concentration and tested on Standard II Nutrient agar seeded with B .subtilis (10 4 colonies forming units/ml) . The linearity of the standard curve in the concentration range 0 .5-100 ug/ml was tested statistically at the 95% confidence level after three replications .
Table I Number of samples analyzed for each experimental group . Each sample of kidney was tested in duplicate . I .p . administration of TBM was performed on GD 10-19 (sperm positive vaginal smear= GD 0) Qualitative screening assay
Semiquantitative assay
Doses of TBM Day from last treatment
1st
Samples
0 30 mg/kg h .w . 60 mg/kg h .w . (Group C) (Group A) (Group B)
30 mg/kg .h .w . (Group S)
Maternal kidney Fetal kidney Placentae
1 7 7
3 35* 37
2 12 12
1 13 13
Maternal kidney
1
3
2
2
Newborn kidney
12
28
26
26
Maternal kidney
1
4
2
6th
Ilth 13 50 20 32 Newborn kidney *Two samples of fetal kidneys in group A (30 mg/kg b .w .) were lost for analysis .
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Tissue assay A qualitative screening assay was performed to verify : (1) the absence of intrinsic inhibitory activity in control samples ; (2) the sensitivity of the plates under our assay conditions ; (3) the presence of TBM residues both in maternal samples (renal and placental tissue) and in litters (fetuses and newborn kidneys) from groups A and B . Afterwards, a Cooper's quantitative assay was performed with the aim of obtaining a semiquantitative evaluation of the residues at 30 mg/kg b .w . dose levels (group S) [10] . With the exception of the placentae, each sample was placed in duplicate in the same plate, against three replicates of the standard concentrations, ranging from 0 .5 to 100 ug/ml (0 .5, 1 .0, 5 .0, 10 .0, 50 .0, 100 .0 µg/ml) . The plates were then incubated upright at 32°C for 24 h .
RESULTS No intrinsic inhibitory activity was present in control samples . The lower limit of sensitivity of the assay was 0 .5 pg/ml . The presence of TBM residues in renal and placental tissues in each litter, as detected by the qualitative screening assay is shown in Fig 1 . At the 1st sampling all three litters of group A contained some 1
a . ---
-
. nd Na xkmaI number • cesarean section • brlh
I∎ x 8 m
u
Nc .
a
placenta
'
® 0 10 toet- kdrey r(0 a
+
3- -
Last aaid` treatment
I
-
nbt
.
k,arey
T
, ~1 f tw
--
ER_~ dam kd,a 40 5m
7h fh ytn
tPh
Fig 1 . Presence of TBM residues (detected by the qualitative screening assay) at the 1st, 6th, 11th day after the last day of treatment (i .e . 1st, 2nd and 3rd sampling, respectively) in placentae and in maternal, fetal and neonatal kidneys of the two groups exposed i .p . to 30 mg/kg b .w . (Group A) . (One sample each of fetal kidney was lost for analysis in litters I and 3 of group A.) and 60 mg/kg b .w . (Group B) of TBM from GD 10 to GD 19 (last day of treatment) . The size of microbial inhibition zone is represented by the boxes : empty = from 0 to 1 mm; half-full = between I and 3 mm ; full = >3 mm .
Pharmacological Research, Vol. 26, No . 3, 1992
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Table II Frequency (positive/total) and percentage (%) of TBM presence according to the qualitative screening assay in fetal and neonatal kidneys of dams treated i .p . with 30 mg/kg b .w . (Group A) and 60 mg/kg b .w. (Group B) according to sex (m = males ; f = females) newborns
fetuses
Day from last treatment Doses
1st
6th
11th
Group A 30 mg/kg %:
m. 5/13 38 .4
f. 9/22 40 .9
0/13 0
f. 0/15 0
1/29 3 .4
f. 3/21 14 .2
Group B 60 mg/kg 17c-
4/4 100 .0
7/8 87 .5
0/15 0
0/11 0
0/10 0
2/10 20 .0
M.
M.
negative samples, and the frequency of placentae showing residues was greater than that of fetal kidneys, while the opposite was apparent in group B . At the 2nd sampling, no newborns with renal residues of TBM were found . At the 3rd sampling, 8% and 10% of newborns showed presence of TBM in groups A and B ., respectively . No statistically significant differences were found between the two sexes with regard to the frequency of the TBM presence at both those levels . However, if the findings at 30 mg/kg b .w . and 60 mg/kg b .w . were pooled, a marginally significantly (P=0 .05, Fisher's exact test) higher frequency was found in females . Cumulative percentage frequencies for each sex at each day of sampling are shown in Table II . Results from the semiquantitative assay showed the renal concentration in the newborns which still had residues are comparable to those observed in fetuses, though a very high presence of TBM was detected in the kidneys of one oedematous fetus . The results from the semiquantitative assay confirm that placental accumulation is more prevalent than renal accumulation at 30 mg/kg b .w . (Table III) .
DISCUSSION The nephrotoxic aminoglycoside TBM did not show any teratogenic or developmentally toxic potential when tested in a conventional test protocol at doses up to 100 mg/kg b .w ./day [4] . On the other hand, specific adverse renal developmental effects have been demonstrated in the Sprague-Dawley rat when 10 i .p . administrations of 30 or 60 mg/kg b .w ./day were given to dams from GD 10 to GD 19 [6] . Those effects can be summarized as dose-related increases of the glomerular density and of the relative medullary area, both suggestive of an
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Pharmacological Research, Vol . 26, No . 3, /992
Table III Semiquantitative assay : concentration (pg/g of sample tissue) of TBM in dams treated i.p . (on GD 10-19) with 30 mg/kg b .w . (Group S) and in their titters (placentae and kidneys) Day from last treatment
1st
6th
Maternal kidney
34
24
Specimen n.
placentae
fetal kidneys
1 2 3
20 20 8
108* 2 -
4 5 6 7 9 10 11
6 4 4 4 4 4 1 -
12 13
-
8
14 15 16 17
29
I I th
20
10
neonatal kidneys 4
-
-
4
1
-
-
-
1 -
1 -
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
18
-
19
-
*Kidney from oedematous fetus .
impaired development of the proximal tubule . These findings were even more apparent in newborns on PD 9 than in fetuses on GD 20 . At both dose levels histopathological changes of the tubules were observed in fetuses and, mainly at the higher dose, in newborns . The present study was conducted to support the histopathological and histomorphometrical findings in the rat, with information about the presence of TBM in the kidneys and placentae of conceptuses : the detection microbiological method was chosen to make the presence of TBM in a biologically active form evident . Aminoglycosides enter the renal tubular cell by adhesion to membrane phospholipids and are subsequently entrapped within lysosomes [11] . The covalently bound fraction does not appear to be quantitatively important or to contribute significantly to the toxic effects . According to Aronoff et al . [12] and Brier et al . [7] a direct relationship exists between aminoglycosides nephrotoxicity and their renal parenchymal concentration assessed by means of a microbiological method . The drug crosses the human placenta and accumulates in the fetal kidney as
Pharmacological Research, Vol . 26, No . 3 . 1992
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well as in the placenta with a placenta :kidney ratio of 1 :5 [5] . Our data show that substantial placental transfer and some placental accumulation occur in the Sprague-Dawley rat . The presence of placental residues of TBM is unlikely to be due to plasmatic TBM in a tissue with a high blood circulation . In fact, the half life of TBM in plasma is short (5 .2 in human fetal serum) [5] and the sampling of the tissue was performed 24 h after the last administration : therefore, some placental uptake of TBM can be assumed . Moreover according to both the qualitative and semiquantitative assay the presence of TBM in placenta seems to be more prevalent than in the fetal kidney at 30 mg/kg b .w . We did not investigate possible microscopical changes in the placenta ; however, delayed development, mortality or malformations other than renal effects were not observed in the fetuses from treated litters . Thus, a gross impairment of the placental function is unlikely . No statistically significant difference was noted in the frequency of TBM residues between male and female conceptuses in either dose group . However, in our previous study, histological effects were noted to be consistently more marked in males, and a difference in response to aminoglycoside nephrotoxicity between sexes has been ascertained in adult rats [13] The semiquantitative TBM assay shows that the renal concentration in dams decreases gradually between days 6 and 11 after the last administration . Overall (i .e . if data from the different experimental groups are pooled), about 6% of the newborns still had detectable amounts on day 6-11 after the last treatment of the dams . However, the frequency of residual compound found in newborns was not obviously related to the in utero exposure or to the time from the last treatment . It is also worth noting that, in the semiquantitative assay, the renal concentrations detected in newborns were comparable to those detected in fetuses . When considering the frequency of newborns still showing renal residues of TBM, one should keep in mind not only the renal accumulation half life of TBM (3 .01 and 11 .00 days, respectively, for 30 day s .c . treatments of 5 and 20 mg/kg b .w .) [7], but also the "dilution effect" induced by the substantial increase of renal tissue (5-6-fold from GD 20 to PD 9, [see 6]) in absence of further exposure to the chemical . Summarizing, biologically active residues of TBM are present in placentae and in fetal kidneys when pregnant rats are administered TBM from GD 10 to GD 19 . Detectable amounts of TBM in kidneys are present in a small proportion of newborns up to, at least, 11 days after the last prenatal exposure . The frequency of such newborns is "scattered" and not obviously related to the level of exposure, to the time elapsed from the end of the exposure and to the sex . It is therefore possible to speculate that they represent a subgroup particularly sensitive to TBM accumulation and/or retention .
REFERENCES 1 . Daston GP, Rehnberg BF, Carver B, Kavlock RJ . Functional teratogens of the rat kidney . Fundam Appl Toxicol 1988 ; 11 : 401-15 .
2 . Gilbert T, Lellevre-Pegorier M, Malienou R, Meulemans M, Merlet-Benichou C . Effects
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of prenatal and postnatal exposure to gentamycin on renal differentiation in the rat . Toxicology 1987 ; 43 : 301-13 . 3 . Chahoud I, Stahlmann R, Merker HJ, Neubert D . Hypertension and nephrotoxic lesions in rats 1 year after prenatal exposure to gentamycin . Arch Toxicol 1989 ; 62 : 274-84 . 4. Welles JS, Emmerson JL, Gibson WR, Nickander R, Owen NV, Anderson RC . Preclinical toxicology studies with Tobramycin . Toxicol Appl Pharmacol 1973 ; 25 : 398-409 . 5 . Bernard B, Garcia-Cazares SJ, Ballard CA, Thrupp LD, Mathies AW, Wehrle PF . Tobramycin : Maternal-fetal pharmacology . Antimicrob Agents Chemother 1977; 11 : 688-94. 6 . Mantovani A, Macri C, Stazi AV, Ricciardi C, Guastadisegni C, Maranghi F . Tobramycin-induced changes in renal histology of fetal and newborns Sprague-Dawley rats . Teratogenesis Carcin Mutagen 1992 (in press) . 7 . Brier ME, Maxer PR, Brier RA, Ilisscher D, Luft FC, Arnoff GR . Relationship between rat renal accumulation of gentamycin, tobramycin, and netilmicin and their nephrotoxicities . Antimicrob Agents Chemother 1985 ; 27 : 812-6 . 8 . Ferrini AM, Aureli P, Ricciardi C, Stazi AV, Macri C, Mantovani A . Renal and placental accumulation of Tobramycin in rat foetuses and newborns exposed in utero . Teratology 1990 ; 42 (2) : 23A-4A . 9 . Levetzow R, Weise E . Zur Ruckstandsuntersuchung Rahmen der amtlichen Fleischuntersuchung . Sclacht- and Viehofztg 1974; 74 : 329-36 . 10. Cooper KE . The theory of antibiotic diffusion zones . In : Kavanagh F, ed . Analytical Microbiology . New York and London : Academic Press, 1972 : 13-30 . 11 . Laurent G, Toubeau G, Heuson-Stiennon JA, Tulkens P, Maldague P . Kidney tissue repair after nephrotoxic injury : biochemical and morphological characterization . CRC Crit Rev Toxicol 1988 ; 19 : 147-83 . 12 . Aronoff GR, Potratz ST, Brier ME, et al . Aminoglycoside accumulation kinetics in rat renal parenchyma . Antimicrob Agents Chemother 1983 ; 23 : 74-8 . 13 . Kourilsky 0, Solez K, Morel-Maroger L, Whelton A, Duhoux P, Sraer JD . The pathology of acute renal failure due to interstitial nephritis in man with comments on the role of interstitial inflammation and sex in gentamycin nephrotoxicity . Medicine 1982 ; 61 : 258-68 .
27 7
PRESENCE OF TOBRAMYCIN IN KIDNEYS AND PLACENTAE OF SPRAGUE-DAWLEY RAT FETUSES AND NEWBORNS FOLLOWING IN UTERO EXPOSURE A . M . FERRINI*, P . AURELI*, C . RICCIARDIt, A . V . STAZIt, C . MACRIt and A . MANTOVANIt *Laboratorio Alimenti and tLaboratorio di Tossicologia Comparata ed Ecotossicologia, Istituto Superiore di Sanita, viale Regina Elena, 299, 00161 Roma, Italy Received in final form I April 1992
SUMMARY The maternal-fetal transfer of tobramycin (TBM) was investigated in the rat by means of a microbiological assay, to assess the presence and amount in the kidneys and placentae of fetuses at gestational day (GD) 20, in the kidneys of newborns 6 and 11 days after the end of treatment and in the kidneys of the dams . In the qualitative assay, pregnant rats were injected i .p . with 0, 30, 60 mg/kg b .w . of TBM on GD 10-19 . A group of dams treated in parallel with 30 mg/kg b .w . was utilized for a microbiological semiquantitative assay . All litters contained some fetuses showing no detectable TBM accumulation in either kidney or in placentae : at 30 mg/kg/b .w . accumulation appeared more prevalent in placenta than in the kidneys of the corresponding fetuses, as confirmed also by the semiquantitative assay . Some newborns (about 6%) of both groups showed detectable renal TBM residues on the 6th and on the 11th day after the end of treatment . The frequency of newborns showing residues was not obviously related to the dose or the day of sampling, and the concentrations of TBM found were comparable to those observed in fetuses . It is possible that they represent a particularly sensitive subgroup . KEY WORDS :
offspring, tobramycin, kidney, placental transfer .
INTRODUCTION The kidney can be a target organ for developmental toxicants, as shown by studies concerning environmental agents [1] or aminoglycoside gentamycin [2,3] . The nephrotoxic aminoglycoside tobramycin (TBM) is apparently devoid of readily detectable adverse developmental effects [4] although it has been demonstrated to be present in the kidneys and placentae of human fetuses from treated mothers [5] . Moreover, TBM elicits microscopical alterations of renal development in SpragueDawley rat fetuses on gestational day (GD) 20 and in newborns on postnatal day 1043-6618/92/070277-08/$08 .00/0
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Pharmacological Research, Vol . 26, No . 3, 1992
(PD) 9, exposed in utero to 60 or 30 mg/kg from GD 10 to GD 19, i .e . the period of major renal organogenesis [6] . The experiments described here were performed to assess the renal and placental accumulation of TBM in rat conceptuses, as well as the persistence of TBM in early post-natal life, following a treatment schedule that was identical to the one leading to micromorphological alterations . The presence and amount of TBM were respectively defined by qualitative and semiquantitative microbiological methods [see, e .g ., 7] to detect biologically active residues . The preliminary results of our study were presented at the 18th European Teratology Society Conference [8] .
MATERIALS AND METHODS Animals
Nulliparous Sprague-Dawley female rats weighing 180-200 g were purchased from Charles River (Calco, Como, Italy) and held for one week for acclimatization . They were kept in a self-contained unit under the following environmental conditions : 22 ± 0 .5 ° C ; humidity 55% ; 12-14 air changes/h ; 12 :12 light/dark photoperiod . Drinking water and food (Open Formula, D .ri Piccioni, Brescia, Italy) were available ad libitum . Compound
Tobramycin (QA179J, batch Y4855, activity : 946 mg/g) was obtained from Eli Lilly Italia S .p .A . (Sesto Fiorentino, Italy) . Doses
Doses were : 30 mg/kg b .w . (group A) ; 60 mg/kg b .w . (group B) ; 30 mg/kg b .w . (group S) of TBM vehicled in a sterile saline solution (I ml solution/300 g b .w .) ; control group (group C), only vehicle solution . Treatment Schedule
The females were mated (2 :1) to adult males and the day of a sperm positive vaginal smear was defined as GD 0 . On GD 10 they were weighed and assigned to the experimental groups . From GD 10 to GD 19 (around 09 .30 a .m . each day) they were treated i .p . to allow a rapid absorption of the drug . On GD 20 (i .e . the day after the last treatment) some of the females in each group were overanaesthetized by i .p . injection of sodium pentothal followed by cervical dislocation . The maternal kidneys, fetal kidneys and placentae were collected in sterile conditions (1st sampling) . Each fetus was sexed, weighed, identified and overanesthetized before sampling . The other dams were allowed to deliver the litters (most of the rats gave birth to their litters on GD 22) . On the 6th and on the 11th day following the last treatment (2nd and 3rd sampling), the dams and their newborns were overanesthetized and killed by cervical dislocation ; the maternal and neonatal kidneys were then collected in sterile conditions . Each newborn was sexed, weighed and identified . The parallel group (group S) received 30 mg/kg b .w ., by the same treatment
Pharmacological Research, Vol. 26, No . 3, 1992
27 9
schedule as above, and the samples were utilized to perform the semiquantitative analysis of TBM residues . For an overview of the experimental design see Table 1 . Since they had been extensively studied in a previous experiment [6] no parameters of maternal or developmental toxicity were monitored in the present study, except cage-side and external observations . All samples were immediately kept at 4°C and assayed within 2 h . Microbiological method
Antibiotic activity was determined by the agar diffusion method with Bacillus subtilis ATCC 6633 (Strain BGA) as the reference strain : this strain is also used in the German Hemmstofftest, because of its sensitivity [9] . Standard curve
The linear concentration range of standard dilutions of TBM was determined according to the standard curve procedure as described by Cooper [10] . An accurately determined amount of TBM was dissolved to give a stock solution containing 1000 ug/ml . From this stock solution, 10 concentrations ranging from 200 to 0 .25 ug/ml were obtained by successive dilutions . Paper disks (13 mm diameter) were soaked with 100 ml of each concentration and tested on Standard II Nutrient agar seeded with B .subtilis (10 4 colonies forming units/ml) . The linearity of the standard curve in the concentration range 0 .5-100 ug/ml was tested statistically at the 95% confidence level after three replications .
Table I Number of samples analyzed for each experimental group . Each sample of kidney was tested in duplicate . I .p . administration of TBM was performed on GD 10-19 (sperm positive vaginal smear= GD 0) Qualitative screening assay
Semiquantitative assay
Doses of TBM Day from last treatment
1st
Samples
0 30 mg/kg h .w . 60 mg/kg h .w . (Group C) (Group A) (Group B)
30 mg/kg .h .w . (Group S)
Maternal kidney Fetal kidney Placentae
1 7 7
3 35* 37
2 12 12
1 13 13
Maternal kidney
1
3
2
2
Newborn kidney
12
28
26
26
Maternal kidney
1
4
2
6th
Ilth 13 50 20 32 Newborn kidney *Two samples of fetal kidneys in group A (30 mg/kg b .w .) were lost for analysis .
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Pharmacological Research, Vol . 26, No . 3, 1992
Tissue assay A qualitative screening assay was performed to verify : (1) the absence of intrinsic inhibitory activity in control samples ; (2) the sensitivity of the plates under our assay conditions ; (3) the presence of TBM residues both in maternal samples (renal and placental tissue) and in litters (fetuses and newborn kidneys) from groups A and B . Afterwards, a Cooper's quantitative assay was performed with the aim of obtaining a semiquantitative evaluation of the residues at 30 mg/kg b .w . dose levels (group S) [10] . With the exception of the placentae, each sample was placed in duplicate in the same plate, against three replicates of the standard concentrations, ranging from 0 .5 to 100 ug/ml (0 .5, 1 .0, 5 .0, 10 .0, 50 .0, 100 .0 µg/ml) . The plates were then incubated upright at 32°C for 24 h .
RESULTS No intrinsic inhibitory activity was present in control samples . The lower limit of sensitivity of the assay was 0 .5 pg/ml . The presence of TBM residues in renal and placental tissues in each litter, as detected by the qualitative screening assay is shown in Fig 1 . At the 1st sampling all three litters of group A contained some 1
a . ---
-
. nd Na xkmaI number • cesarean section • brlh
I∎ x 8 m
u
Nc .
a
placenta
'
® 0 10 toet- kdrey r(0 a
+
3- -
Last aaid` treatment
I
-
nbt
.
k,arey
T
, ~1 f tw
--
ER_~ dam kd,a 40 5m
7h fh ytn
tPh
Fig 1 . Presence of TBM residues (detected by the qualitative screening assay) at the 1st, 6th, 11th day after the last day of treatment (i .e . 1st, 2nd and 3rd sampling, respectively) in placentae and in maternal, fetal and neonatal kidneys of the two groups exposed i .p . to 30 mg/kg b .w . (Group A) . (One sample each of fetal kidney was lost for analysis in litters I and 3 of group A.) and 60 mg/kg b .w . (Group B) of TBM from GD 10 to GD 19 (last day of treatment) . The size of microbial inhibition zone is represented by the boxes : empty = from 0 to 1 mm; half-full = between I and 3 mm ; full = >3 mm .
Pharmacological Research, Vol. 26, No . 3, 1992
28 1
Table II Frequency (positive/total) and percentage (%) of TBM presence according to the qualitative screening assay in fetal and neonatal kidneys of dams treated i .p . with 30 mg/kg b .w . (Group A) and 60 mg/kg b .w. (Group B) according to sex (m = males ; f = females) newborns
fetuses
Day from last treatment Doses
1st
6th
11th
Group A 30 mg/kg %:
m. 5/13 38 .4
f. 9/22 40 .9
0/13 0
f. 0/15 0
1/29 3 .4
f. 3/21 14 .2
Group B 60 mg/kg 17c-
4/4 100 .0
7/8 87 .5
0/15 0
0/11 0
0/10 0
2/10 20 .0
M.
M.
negative samples, and the frequency of placentae showing residues was greater than that of fetal kidneys, while the opposite was apparent in group B . At the 2nd sampling, no newborns with renal residues of TBM were found . At the 3rd sampling, 8% and 10% of newborns showed presence of TBM in groups A and B ., respectively . No statistically significant differences were found between the two sexes with regard to the frequency of the TBM presence at both those levels . However, if the findings at 30 mg/kg b .w . and 60 mg/kg b .w . were pooled, a marginally significantly (P=0 .05, Fisher's exact test) higher frequency was found in females . Cumulative percentage frequencies for each sex at each day of sampling are shown in Table II . Results from the semiquantitative assay showed the renal concentration in the newborns which still had residues are comparable to those observed in fetuses, though a very high presence of TBM was detected in the kidneys of one oedematous fetus . The results from the semiquantitative assay confirm that placental accumulation is more prevalent than renal accumulation at 30 mg/kg b .w . (Table III) .
DISCUSSION The nephrotoxic aminoglycoside TBM did not show any teratogenic or developmentally toxic potential when tested in a conventional test protocol at doses up to 100 mg/kg b .w ./day [4] . On the other hand, specific adverse renal developmental effects have been demonstrated in the Sprague-Dawley rat when 10 i .p . administrations of 30 or 60 mg/kg b .w ./day were given to dams from GD 10 to GD 19 [6] . Those effects can be summarized as dose-related increases of the glomerular density and of the relative medullary area, both suggestive of an
28 2
Pharmacological Research, Vol . 26, No . 3, /992
Table III Semiquantitative assay : concentration (pg/g of sample tissue) of TBM in dams treated i.p . (on GD 10-19) with 30 mg/kg b .w . (Group S) and in their titters (placentae and kidneys) Day from last treatment
1st
6th
Maternal kidney
34
24
Specimen n.
placentae
fetal kidneys
1 2 3
20 20 8
108* 2 -
4 5 6 7 9 10 11
6 4 4 4 4 4 1 -
12 13
-
8
14 15 16 17
29
I I th
20
10
neonatal kidneys 4
-
-
4
1
-
-
-
1 -
1 -
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
18
-
19
-
*Kidney from oedematous fetus .
impaired development of the proximal tubule . These findings were even more apparent in newborns on PD 9 than in fetuses on GD 20 . At both dose levels histopathological changes of the tubules were observed in fetuses and, mainly at the higher dose, in newborns . The present study was conducted to support the histopathological and histomorphometrical findings in the rat, with information about the presence of TBM in the kidneys and placentae of conceptuses : the detection microbiological method was chosen to make the presence of TBM in a biologically active form evident . Aminoglycosides enter the renal tubular cell by adhesion to membrane phospholipids and are subsequently entrapped within lysosomes [11] . The covalently bound fraction does not appear to be quantitatively important or to contribute significantly to the toxic effects . According to Aronoff et al . [12] and Brier et al . [7] a direct relationship exists between aminoglycosides nephrotoxicity and their renal parenchymal concentration assessed by means of a microbiological method . The drug crosses the human placenta and accumulates in the fetal kidney as
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well as in the placenta with a placenta :kidney ratio of 1 :5 [5] . Our data show that substantial placental transfer and some placental accumulation occur in the Sprague-Dawley rat . The presence of placental residues of TBM is unlikely to be due to plasmatic TBM in a tissue with a high blood circulation . In fact, the half life of TBM in plasma is short (5 .2 in human fetal serum) [5] and the sampling of the tissue was performed 24 h after the last administration : therefore, some placental uptake of TBM can be assumed . Moreover according to both the qualitative and semiquantitative assay the presence of TBM in placenta seems to be more prevalent than in the fetal kidney at 30 mg/kg b .w . We did not investigate possible microscopical changes in the placenta ; however, delayed development, mortality or malformations other than renal effects were not observed in the fetuses from treated litters . Thus, a gross impairment of the placental function is unlikely . No statistically significant difference was noted in the frequency of TBM residues between male and female conceptuses in either dose group . However, in our previous study, histological effects were noted to be consistently more marked in males, and a difference in response to aminoglycoside nephrotoxicity between sexes has been ascertained in adult rats [13] The semiquantitative TBM assay shows that the renal concentration in dams decreases gradually between days 6 and 11 after the last administration . Overall (i .e . if data from the different experimental groups are pooled), about 6% of the newborns still had detectable amounts on day 6-11 after the last treatment of the dams . However, the frequency of residual compound found in newborns was not obviously related to the in utero exposure or to the time from the last treatment . It is also worth noting that, in the semiquantitative assay, the renal concentrations detected in newborns were comparable to those detected in fetuses . When considering the frequency of newborns still showing renal residues of TBM, one should keep in mind not only the renal accumulation half life of TBM (3 .01 and 11 .00 days, respectively, for 30 day s .c . treatments of 5 and 20 mg/kg b .w .) [7], but also the "dilution effect" induced by the substantial increase of renal tissue (5-6-fold from GD 20 to PD 9, [see 6]) in absence of further exposure to the chemical . Summarizing, biologically active residues of TBM are present in placentae and in fetal kidneys when pregnant rats are administered TBM from GD 10 to GD 19 . Detectable amounts of TBM in kidneys are present in a small proportion of newborns up to, at least, 11 days after the last prenatal exposure . The frequency of such newborns is "scattered" and not obviously related to the level of exposure, to the time elapsed from the end of the exposure and to the sex . It is therefore possible to speculate that they represent a subgroup particularly sensitive to TBM accumulation and/or retention .
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