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In vivo and in vitro effects of azaconazole on renal function in the Fischer 344 rat
Toxicology, 34 (1985) 1--11
Elsevier Scientific Publishers Ireland Ltd.
IN VIVO A N D IN V I T R O E F F E C T S O F A Z A C O N A Z O L E ON R E N A L F U N C T I O N IN T H E F I S C H E R 3 4 4 R A T
GARY O, RANKIN a, DAVID J. YANG a, KIM CRESSEY-VENEZIANO a, RUU T. WANG b and PATRICK I. BROWN b Departments o f Pharmacology a and Anatomy b, Marshall University School o f Medicine, Huntington, WV 25701 (U.S.A.)
(Received July 5th, 1984) (Accepted September 21st, 1984)
SUMMARY Azacon~azole is an e x p e r i m e n t a l agricultural fungicide which has s h o w n p r o m i s e f o r use in c o n t r o l l i n g p o w d e r y m i l d e w o n crops and bean rust. This s t u d y e x a m i n e d t h e n e p h r o t o x i c p o t e n t i a l o f a single i n t r a p e r i t o n e a l azaconazole i n j e c t i o n (0.4 or 0.6 m m o l / k g ) or daily a z a c o n a z o l e a d m i n i s t r a t i o n (0.1 or 0.3 m m o l / k g / d a y ) f o r 7 d a y s in male Fischer 3 4 4 rats. T h e in vitro effects o f a z a c o n a z o l e o n t h e a c c u m u l a t i o n o f organic ions b y renal cortical slices also were e x a m i n e d . A c u t e a z a c o n a z o l e a d m i n i s t r a t i o n (0.4 or 0.6 m m o l / k g , i.p.) p r o d u c e d a m a r k e d decrease in urine v o l u m e at 6 h. By 48 h urine v o l u m e was still decreased in t h e 0 . 6 - m m o l / k g g r o u p b u t n o t t h e 0 . 4 - m m o l / k g group. P r o t e i n u r i a (+ + ) and slight h e m a t u r i a were seen in t h e 0 . 6 - m m o l / k g g r o u p o n b o t h t r e a t m e n t days. A c c u m u l a t i o n o f p - a m i n o h i p p u r a t e (PAH) b y renal cortical slices was s t i m u l a t e d in b o t h a z a c o n a z o l e - t r e a t e d groups while t e t r a e t h y l a m m o n i u m {TEA) a c c u m u l a t i o n was n o t altered. No changes in b l o o d u r e a n i t r o g e n c o n c e n t r a t i o n , k i d n e y weight or renal m o r p h o l o g y were p r o d u c e d at 48 h p o s t i n j e c t i o n by e i t h e r a z a c o n a z o l e dose. Daily administ r a t i o n o f a z a c o n a z o l e (0.1 o r 0.3 m m o l / k g / d a y ) did n o t significantly alter a n y o f t h e renal p a r a m e t e r s studied. I n c u b a t i o n o f renal cortical slices with increasing c o n c e n t r a t i o n s o f a z a c o n a z o l e f r o m 10 -5 M to 10 -3 M caused a c o n t i n u e d r e d u c t i o n in T E A a c c u m u l a t i o n . PAH a c c u m u l a t i o n was decreased significantly following i n c u b a t i o n with a z a c o n a z o l e 10 -s or 10 -3 M. These results indicate t h a t a z a c o n a z o l e is capable of p r o d u c i n g a c u t e , reversible renal e f f e c t s at doses equal t o or less t h a n 0.6 m m o l / k g and altering organic ion t r a n s p o r t b o t h in vivo and in vitro. T h e lack o f m a r k e d renal effects
Abbreviations: BUN, blood urea nitrogen; H and E, hematoxylin and eosin; PAH, p-
aminohippurate; PAS, periodic acid Schiff; TEA, tetraethylammonium. 0300-483X/85/$03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
following exposure to azaconazole is favorable for the development of this c o m p o u n d as an agricultural fungicide.
Key words: Fungicides; Triazoles; hydrocarbons
Nephrotoxicity;
Rats;
Halogenated
INTRODUCTION The search for new, potent antifungal agents has resulted in the discovery of several promising fungicidal compounds. However, many of these compounds have been shown to be nephrotoxic in man or animal studies. For example, N-(3,5
N~N..PCl C~CI 0 /
"0--
I
I
AZACONAZOLE Fig. 1, Chemical structure of azaconazo|e (R 28644).
2
vivo and in vitro effects of azaconazole on renal function were examined. Renal tissue was examined for changes in morphology using light microscopy. METHODS Animals
Male Fischer 344 rats (200--300 g) were used in all experiments. Rats were purchased from Hilltop Lab Animals, Inc. (Scottdale, PA) and housed in rooms with controlled temperature (21--23°C), humidity and light (12 h light/12 h dark cycle). Rats were housed singly in stainless steel metabolism cages during the in vivo experiments. At all other times rats were housed in groups of not more than 6 rats per group in standard plastic animal cages. In vivo studies
Three days prior to their placement in metabolism cages, each rat had a blood sample taken from the tail for the determination of the basal blood urea nitrogen concentration (BUN, Sigma Kit No. 640). Rats were allowed free access to food (powdered Purina R o d e n t Chow) and water on the first day of individual housing. The next day (control day), food was removed around 0900 h and withheld for 6 h to assure collection of a urine specimen without food contamination. The 6-h urine specimens were analyzed qualitatively (Multistix ®, Ames Company) for the presence of protein, glucose, ketones and blood, and the results served as that day's urine content analysis. Body weight, food and water intake, and urine volume and osmolality were determined at 24-h intervals. Following the control day, rats (4 rats/group) in the acute studies received a single injection of vehicle only (sesame oil, 2.5 ml/kg, i.p.) or azaconazole (0.4, 0.6, or 1.0 mmol/kg, i.p.) at 0900 h and monitored as described above. At 48 h postinjection a blood sample was taken from the tail of each rat for BUN concentration determinations, and the rats were killed by cervical dislocation. Both kidneys were removed rapidly with the left kidney being used for the renal slice studies. The right kidney was weighed and examined for histological changes as described below. No animals died in the vehicle only groups or azaconazole 0.4- or 0.6-mmol/kg groups prior to sacrifice. However, all rats died in the azaconazole 1.0-mmol/kg group before 48 h, and their data are not included. In the subacute studies, rats (4 rats/group) received a daily i.p. injection of azaconazole (0.1 or 0.3 mmol/kg) or vehicle for 7 days. Urinary parameters, food and water intake, and b o d y weight were measured daily. BUN concentration, kidney weights, and organic ion accumulation by renal cortical slices were measured on day 7 as described in the acute studies. No animals died in any treatment or control group. Control rats were pair-fed for their appropriate treatment groups. This assured that any observed renal response was due to azaconazole treatment and not due to decreased food intake.
In vitro studies
In these studies rats were killed by cervical dislocation around 0900 h, and renal slices were prepared and processed as described below. Tissue slices were incubated with azaconazole for 30 min. [14C] PAH or [~4C] TEA were added, and the tissue incubation continued for 90 rain. Accumulation of PAH and TEA was determined as described below for the renal slice studies. Azaconazole solutions were prepared in 95% ethanol such that addition of 30/11 of the azaconazole solution would yield the desired final azaconazole concentration in the 3 ml of incubation medium. Each control sample had 30 t~l of 95% ethanol only added to the incubation medium. R e n a l slice studies
All rats were killed by cervical dislocation at the appropriate time. Kidneys were removed rapidly and placed in ice-cold Krebs-Ringer phosphate buffer (pH 7.4) containing 5.0 mM potassium and 1.0 mM calcium. Renal cortical slices were prepared freehand, and approximately 100 mg of tissue were incubated with shaking for 90 min in the same buffer at 25°C under a 100% oxygen atmosphere in a D u b n o f f metabolic shaker. Sodium lactate (10 -2 M) was used in some experiments. Following the incubation period, tissues were removed from the bathing media, blotted, weighed and homogenized in distilled water (4 ml) using a Tekmar tissumizer. Aliquots (1-ml) of the homogenates were placed in liquid scintillation counting vials and mixed with 9 ml of PCS counting solution. The incubation media were counted in a similar manner using 0.5-ml aliquots of media and 9.5 ml of PCS. Counting was performed using a Beckman LS-9000 liquid scintillation spectrometer with external standard quench correction. The uptake of p-['4C]aminohippurate (PAH) and ['4C]tetraethylammonium (TEA) was studied. For each compound, enough radioactive compound was added to yield about 0.02 #Ci of 14C/ml of incubation medium and sufficient unlabelled material to yield an initial concentration of 7 × 10 -S M for PAH and 10 -S M for TEA. Isotopes were obtained from New England Nuclear Corporation, Boston, MA. Accumulation of PAH and TEA by renal cortical slices was expressed as the slice to medium (S/M) ratio where S equaled the radioactivity (dpm)/g of tissue and M equaled the radioactivity (dpm)/ml of incubation medium. His t o logy
Following cervical dislocation, the right kidney was removed rapidly, weighed, cut in half lengthwise and placed in 10% neutral buffered formalin (3.7% formaldehyde in 75 mM sodium phosphate buffer, pH 7.0). Fixed tissues were embedded in paraffin, sectioned at 6--8 ~m and stained with hematoxylin and eosin (H and E) or periodic acid Schiff (PAS) reagent before histological examination. Sections of cortex, medulla and papilla were prepared and examined for histopathology using light microscopy (EPOI Lab I microscope). Tissues from treated animals were compared to tissues from pair-fed controls for the presence of chemically-induced changes in morphology.
Statistical analysis All data were analyzed using one-way analysis of variance and/ or Student's t-test. RESULTS
Acute in vivo experiments Azaconazole administration (0.4 or 0.6 mmol/kg) reduced food intake (P < 0.05) on bot h t r e a t m e n t days (Table I). To assure t hat any changes in renal f u n ction we observed were not due to decreased food intake, pair-fed c o n t r o l groups were used. The decrease in f o o d intake was accompanied by decreased water intake (Fig. 2). Water intake was n o t different between the azaconazole 0.4 m m o l / k g group and its pair-fed control group on any day, while the azaconazole 0.6-mmol/kg group drank significantly less water than their pair-fed c o n t r o l group on t r e a t m e n t day 1 but not on t r e a t m e n t day 2. b r i n e volume was markedly reduced at the 6-h measurement on both t r e a t m e n t days by azaconazole (0.4 or 0.6 mmol/kg). On t r e a t m e n t day 1 no urine was p r o d u c e d at 6 h in the 0.4-mmol/kg t r e a t m e n t group, and only 1 rat had voided urine, in the 0.6-mmol/kg t r e a t m e n t group. On day 2 urine volume was not significantly reduced in t he 0.4-mmol/kg group at 6 h but remained decreased in the 0.6-mmol/kg group. The 24-h urine volume was reduced b y azaconazole (0.4 or 0.6-mmol/kg) t r e a t m e n t on day 1. On day 2 the 24-h urine o u t p u t o f the 0.4-mmol/kg t r e a t m e n t group was not significantly reduced, but t he 0.6-mmol/kg t r e a t m e n t group still had significantly reduced urine o u t p u t . Urine volume was not altered in pair-fed control rats at th e 6-h or 24-h measurements on either t r e a t m e n t day. Urine osmolality was decreased in all groups on t r e a t m e n t day 1 when c o m p a r e d to appropriate p r e t r e a t m e n t values and in all groups except the 0.6-mmol/kg pair-fed c o n t r o l group on t r e a t m e n t day 2 (Fig. 2). On day 1, the decrease in urinary osmolality seen in t he 0.6-mmol/kg t r e a t m e n t group was significantly larger (P < 0.05) than the decrease in urinary osmolality TABLE I E F F E C T O F A Z A C O N A Z O L E ON F O O D I N T A K E a A z a c o n a z o l e dose (mmol/kg)
0.4 0.6
F o o d intake (g)b day0
day]
day2
18.14-1.5 18.04-1.2
4.5±0.6 c 1.44-0.6 c
11.64-0.4 c 9.54-2.1 c
aValues are m e a n s 4- S.E. for N = 4 rats. b F o o d intake was m e a s u r e d at 24-h intervals for t h e c o n t r o l day ( d a y 0) and at 24 h (day 1) a n d 48 h (day 2) p o s t i n j e c t i o n . c s i g n i f i c a n t l y d i f f e r e n t f r o m t h e day 0 value, P < 0.05.
5
c ~ 0-4mmol/kg ; control : v. 0-4mmol/kg ;treated 0-6m mol/kg ;control -" "- O'6mmol/kl ;treated
5-
15
E E • -,a
E
lO-
3
&.e,J Z 2i
~--
S
l-
DAYSAFTERTREATMENT
DAYSAFTERTREATMENT 30~0~ f
40EE 30 Z
*
~"
2000
~
]~
20-
10-
o
i
i
DAYSAFTERTREATMENT
DAYSAFTERTREATMENT
Fig. 2. E f f e c t o f a z a c o n a z o l e o n u r i n e v o l u m e , w a t e r i n t a k e a n d u r i n e o s m o l a l i t y . E a c h v a l u e is t h e m e a n a n d t h e v e r t i c a l line t h e s t a n d a r d e r r o r for N = 4. S o m e g r o u p s o f r a t s ( t r e a t e d ) w e r e a d m i n i s t e r e d a s i n g l e i.p. i n j e c t i o n o f a z a c o n a z o l e at t h e d o s e i n d i c a t e d while other groups (control) received vehicle only and were pair-fed for the appropriate t r e a t e d g r o u p . A n a s t e r i s k i n d i c a t e s P < 0 . 0 5 w h e n c o m p a r e d to t h e p r e t r e a t m e n t v a l u e for t h e p a r t i c u l a r g r o u p .
observed in t h e c o r r e s p o n d i n g pair-fed c o n t r o l g r o u p . T h e decreases observed in urinary o s m o l a l i t y in t r e a t e d groups o n day 2 were n o t d i f f e r e n t f r o m those seen in the a p p r o p r i a t e c o n t r o l groups. Urine c o n t e n t was n o t m a r k e d l y altered b y a z a c o n a z o l e . In t h e a z a c o n a z o l e 0 . 4 - m m o l / k g group, no urine was p r o d u c e d b y 6 h o n t r e a t m e n t d a y 1 so t h a t n o m e a s u r e m e n t s c o u l d be o b t a i n e d . On t r e a t m e n t d a y 2 o n l y h e m a t u r i a (trace) was n o t e d in this group. In t h e a z a c o n a z o l e 0 . 6 - m m o l / k g group, p r o t e i n u r i a ( + + ) and h e m a t u r i a (trace) were d e t e c t e d on b o t h t r e a t m e n t days. No urinary c o n t e n t changes were n o t e d in either pair-fed c o n t r o l group.
6
The BUN concentration in treated animals was not increased at 48 h by either dose of azaconazole. In addition, kidney weight at 48 h was not different between either treatment group and its pair-fed control. The accumulation of PAH by renal cortical slices was increased by azaconazole when compared to PAH accumulation by cortical slices from control rats, but TEA accumulation was not altered by azaconazole administration (Fig. 3). Basal PAH uptake was increased by azaconazole 0.4 mmol/kg, and lactate-stimulated PAH uptake was increased in both azaconazole treatment groups at 48 h postinjection. Increasing the dose of azaconazole to 1.0 mmol/kg resulted in the death of all treated rats by 30 h. Two animals died on treatment day 1 between 6 h and 24 h. The 2 animals surviving the first 24 h produced 0.6 and 2.0 ml/24 h. respectively. These remaining animals died on treatment day 2 before the 6-h urinary measurements. All rats were anuric at 6 h on treatment day 1, so that urine content measurements could not be performed, and eventually died in acute renal failure. Examination of renal tissue from azaconazole-treated rats at 48 h postinjection by light microscopy did not reveal any significant changes in renal morphology when compared to renal tissue from the appropriate pair-fed control group. Subacute in vivo experiments Since acute azaconazole administration (0.4 or 0.6 mmol/kg) appeared to produce only mild renal effects, it was felt that reducing the azaconazole dose and administering it daily (0.1 or 0.3 mmol/kg/day) for 7 days might [ " 0"4 m mol/kg'day ; control L~ 0-4 m mol/kg/day: treated
25
[ ] 0'6 m mol,'kg,,'day: control m o . G m mol/kg/day:treated 20 0
~-. 15 cC
10-
5
i
PAH
i
i
PAH + LACTATE
TEA
Fig. 3. E f f e c t o f a z a c o n a z o l e o n p - a m i n o h i p p u r a t e ( P A H ) a n d t e t r a e t h y l a m m o n i u m (TEA) a c c u m u l a t i o n by renal cortical slices. S o m e groups o f rats ( t r e a t e d ) were given a single i.p. i n j e c t i o n o f a z a c o n a z o l e at t h e dose indicated while o t h e r groups ( c o n t r o l ) received vehicle o n l y a n d were pair-fed for t h e a p p r o p r i a t e t r e a t e d group. K i d n e y s used in t h e s t u d y were o b t a i n e d at 48 h p o s t - i n j e c t i o n . T h e height o f each bar is t h e m e a n for N = 4. An asterisk indicates P < 0.05 w h e n c o m p a r e d to pair-fed controls.
7
20-
I.-,-
-" --
-" --
PAH PAH + LACTATE
-"
--
TEA
IS-
()
1026M 1025M 1024M lO-'3M
AZACONAZOLECONCENTRATION Fig. 4. In vitro effects o f a z a c o n a z o l e o n p - a m i n o h i p p u r a t e (PAH) and t e t r a e t h y l a m m o n i u m ( T E A ) a c c u m u l a t i o n b y renal cortical slices. Each value r e p r e s e n t s the m e a n and t h e vertical line the s t a n d a r d error for N = 4 - 6 e x p e r i m e n t s . A n asterisk indicates P < 0.05 w h e n c o m p a r e d to c o n t r o l e x p e r i m e n t s .
reveal wh ethe r or not exposure to azaconazole produces n e p h r o t o x i c i t y in rats. Although f o o d intake was slightly but significantly reduced for 2 days {data not shown) in the azaconazole 0.1-mmol/kg/day group, the renal parameters studied were not altered. Likewise, azaconazole 0.3 m m o l / k g / d a y administration did n o t consistently alter renal function. BUN concentration, kidney weight and organic ion accumulation by renal cortical slices were not different among t he azaconazole t r e a t e d (0.1 or 0.3 m m o l / k g / d a y ) and their pair-fed co n t r ol groups. Renal m o r p h o l o g y was not altered in either treatmerit group when c o m p a r e d to c ont r ol animals. In vitro experiments Renal cortical slices incubated with azaconazole at various concentrations exhibited different tendencies for accumulating the organic anion PAH and the organic cation T E A (Fig. 4). Basal and lactate-stimulated PAH and T E A accumulations steadily declined as the azaconazole c o n c e n t r a t i o n in the incubation media increased. All 3 transport parameters were decreased significantly in t he presence of 10 -s M azaconazole, but basal and lactatestimulated PAH accumulation increased to cont rol levels in bathing media containing 10-4 M azaconazole. With a c o n c e n t r a t i o n o f 10 -3 M azaconazole in the media bot h basal and lactate-stimulated PAH accumulations were again significantly decreased. DISCUSSION
The results of this study d e m o n s t r a t e t hat azaconazole can p r o d u c e reversible effects on renal parameters in rats following acute administration of nonlethal doses and alter organic ion accumulation by renal cortical slices following in vivo or in vitro exposure.
8
The production of oliguria or anuria in rats treated acutely with azaconazole was rapid in onset and reversible at doses of 0.6 mmol/kg or lower. Similar reversible renal dysfunction has been reported following administration of therapeutically useful antifungal agents [12--15] or exposure to agricultural fungicides [16]. However, renal dysfunction may contribute to death at lethal azaconazole concentrations, since all rats in the 1.0mmol/kg dose group died in acute renal failure. Although urinary osmolality decreased following azaconazole administration, it is unlikely that this is a primary effect of azaconazole on the kidney. Both pair-fed control groups also excreted a more dilute urine on treatment day 1, and the control group for the 0.4-mmol/kg treatment group exhibited decreased urinary osmolality on treatment day 2. These observations emphasize the importance of using pair-fed control groups for studies involving compounds which markedly depress food intake. Organic ion transport was alerted by azaconazole after in vivo or in vitro exposure. However, the extent of organic ion accumulation varied depending upon the m e t h o d of exposure to azaconazole, azaconazole dose or concentration, and the organic ion studied. In the in vivo experiments, PAH accumulation was stimulated at 48 h postinjection, while TEA accumulation was not altered. Similar increases in PAH uptake without altering organic cation accumulation have been observed following administration of gentamicin [17], pentobarbital [18], potassium dichromate [19,20] or trish y d r o x y m e t h y l a m i n o m e t h a n e [21]. In addition, a number of metabolic intermediates of the glycolytic and Krebs pathways stimulate both PAH and TEA transport with PAH transport being enhanced to a much greater extent [22--24]. The reasons for this difference in the accumulation of organic anions and cations is not known with certainty. These effects might be related to an interaction of the test c o m p o u n d with specific sites in the proximal tubules [19] or to a decreased efflux of accumulated organic ion [251. Pretreatment of renal cortical slices in vitro with azaconazole resulted in a decline in PAH (basal and lactate-stimulated) and TEA accumulation. Return of PAH accumulation to control S/M ratios in the presence of 10 -~ M azaconazole probably reflects the variability of the small sample size. The marked decrease in both organic anion and cation accumulation at 10 -3 M azaconazole suggests a generalized toxic effect on all segments of the proximal tubules. Although azaconazole produces acute renal effects in rats, other experimental agricultural fungicides have been shown to produce more severe renal dysfunction. For example, a single 0.4 mmol/kg, i.p. injection of N-(3,5-dichlorophenyl)succinimide to Sprague--Dawley rats produces marked diuresis, proteinuria, glucosuria and hematuria, elevates BUN concentration, decreases accumulation of PAH and TEA by renal cortical slices and produces acute tubular necrosis [4]. Similar results are obtained in Fischer 344 rats (unpublished results). Administration of azaconazole (0.4 mmol/kg, i.p.) produced oliguria but did not elevate BUN, decrease PAH or TEA accumu-
lation or alter renal m or phol ogy. Thus, azaconazole does not produce the severity o f renal effects seen with some experimental fungicides. In summary, azaconazole is an experimental agricultural fungicide capable of producing acute, reversible renal effects at doses equal to or less than 0.6 mmol/kg. Organic ion transport is altered bot h in vivo and in vitro, but the mechanisms producing these changes are not clear. The lack of marked renal d y s f u n c t i o n following subacute exposure or altered renal m o r p h o l o g y after acute or subacute exposure suggests that any long lasting effects on renal function p r oduc e d by azaconazole are probably minor. Therefore, azaconazole might prove to be safer as an agricultural fungicide than other compounds currently being developed, since unlike dimetachlone and some ot her experimental fungicides, azaconazole does not appear to be n e p h r o t o x i c following acute or subacute exposure in rats. ACKNOWLEDGEMENTS
Th e authors would like to thank Janssen Pharmaceutica, Inc. for their generous gift of azaconazole. T he authors also would like to thank E. Patricia Lahoda and Hermandra Shah for their excellent technical assistance. REFERENCES I A. Fujinami, T. Ozaki and S. Yamamoto, Studies on biological activity of cyclic imide compounds. Part I. Antimierobial activity of 3-phenyloxazolidine-2,4-diones and related compounds. Agric. Biol. Chem., 35 (1971) 1707. 2 A. Fujinami, T. Ozaki, K. Nodera and K. Tanaka, Studies on biological activity of cyclic imide compounds. Part II. Antimicrobial activity of 1-phenylpyrrolidine-2,5diones and related compounds. Agric. Biol. Chem., 36 (1972) 318. 3 A. Fujinami, N. Tottri, T. Kato and N. Kameda, Antimierobial activity of 3-phenyloxazolidine-2,4-diones and related compounds. Agrie. Biol. Chem., 36 (1972) 1623. 4 G.O. Rankin, Nephrotoxicity following acute administration of N-(3,5-diehlorophenyl)suceinimide in rats. Toxicology, 23 (1982) 21. 5 G.O. Rankin, K. Cressey-Veneziano and P.I. Brown, Onset of and recovery from acute N-(3,5-diehlorophenyl)succinimide-induced nephrotoxieity in Sprague--Dawley rats. Toxicology, 30 (1984) 205. 6 S. Sugihara, Y. Shinohara, Y. Miyata, K. Inoue and N. Ito, Pathologic analysis of chemical nephritis in rats induced by N-(3,5-dichlorophenyl) succinimide. Lab. Invest., 33 (1975) 219. 7 E.F. Godefroi, d. Heeres, J. Van Cutsem and P.A.J. Janssen, The preparation and antimyeotic properties of derivatives of 1-phenethylimidazole. J. Med. Chem., 12 (1969) 784. 8 J. Heeres, L.J.J. Baekx and J. Van Cutsem, Synthesis and antimycotic properties of 1-(2-alkyl-2-phenylethyl)-lH-imidazoles. J. Med. Chem., 19 (1976) 1148. 9 J. Van Cutsem and D. Thienpont, Miconazole, a broad spectrum antimyeotic agent with antibacterial activity. Chemotherapy (Basel), 17 (1972) 392. 10 D.H. Bartlett and N . E Ballard, The effectiveness of Gauzatine and Imazalil as seed treatment fungicides in barley. Proc. Br. Insectic. Fungic. Conf., 8th, 1 (1975) 205. 11 J. Van Gestel, J. Heeres, M. danssen and G. Van Reet, Synthesis and screening of a new group of fungicides: 1-(2-Phenyl-l,3-dioxolan-2-ylmethyl)-l,2,4-triazoles. Pestle. Sei., 11 (1980) 95.
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12 F.J. Takacs, A.M. Tomkiewiez and J.P, Merrill, Amphotericin B nephrotoxicity with irreversible renal failure. Ann. Intern. Med., 59 (1963) 716. 13 T.H. Gouge and V.T. Andriole, An experimental model of amphotericin B nephrotoxicity with renal tubular acidosis. J. Lab. Clin. Med., 78 (1971) 713. 14 J.L. Burgess and R. Birchall, Nephrotoxicity of amphotericin B, with emphasis on changes in tubular function. Am. J. Med., 53 (1972) 77. 15 H.B. Niell, Miconazole carrier solution, hyperlipidaemia and hematologic problems. N. Engl. J. Med., 296 (1977) 1479. 16 A. Koizum!, S. Shiojima, M. Omiya, S. Nakano, N. Sato and M. Ikeda, Acute renal failure and maneb (manganous ethylenebis [dithiocarbamate])exposure. J. Am. Med. Assoc., 242 (1979) 2583. 17 L. Cohen, R. Lapkin and G.J. Kaloyanides, Effect of gentamicin on renal function in the rat. J. Pharmacol. Exp. Ther., 193 (1975) 264. 18 W.O. Berndt and I.K. Ho, Effects of morphine and pentobarbital on mouse and rat renal transport systems. Toxicol. Appl. Pharmacol., 48 (1979) 263. 19 G.H. Hirsch, Differential effects of nephrotoxic agents on renal organic ion transport and metabolism. J. Pharmacol., Exp. Ther., 186 (1973) 593. 20 W.O. Berndt, The effect of potassium dichromate on renal tubular transport processes. Toxicol. Appl. Pharmacol., 32 (1975) 40. 21 D.G. Pegg, K.M. McCormack and J.B. Hook, Effect of substrate pretreatment on renal organic ion transport in the adult rat. Experientia, 32 (1976) 1315. 22 R.J. Cross and J.V. Taggart, Renal tubular transport: Accumulation of p-aminohippurate by rabbit kidney slices. Am. J. Physiol., 161 (1950) 18]. 23 A. Farah, M. Frazer and E. Porter, Studies on the uptake of N'-methylnicotinamide by renal slices of the dog. J. Pharmacot. Exp. Ther., 126 {1959) 202. 24 A. Farah and B. Rennick, Studies on the renal tubular transport of tetraethylammonium ion in renal slices of the dog. J. Pharmacol. Exp. Ther., 117 (1956) 478. 25 G.H. Hirsch and A.P. Pakuts, Renal cortical slice uptake and runout of N~methylnicotinamide and p-aminohippurate after potassium dichromate treatment. Can. J. PhysioL. Pharmacol., 52 (1974) 465.
11
Elsevier Scientific Publishers Ireland Ltd.
IN VIVO A N D IN V I T R O E F F E C T S O F A Z A C O N A Z O L E ON R E N A L F U N C T I O N IN T H E F I S C H E R 3 4 4 R A T
GARY O, RANKIN a, DAVID J. YANG a, KIM CRESSEY-VENEZIANO a, RUU T. WANG b and PATRICK I. BROWN b Departments o f Pharmacology a and Anatomy b, Marshall University School o f Medicine, Huntington, WV 25701 (U.S.A.)
(Received July 5th, 1984) (Accepted September 21st, 1984)
SUMMARY Azacon~azole is an e x p e r i m e n t a l agricultural fungicide which has s h o w n p r o m i s e f o r use in c o n t r o l l i n g p o w d e r y m i l d e w o n crops and bean rust. This s t u d y e x a m i n e d t h e n e p h r o t o x i c p o t e n t i a l o f a single i n t r a p e r i t o n e a l azaconazole i n j e c t i o n (0.4 or 0.6 m m o l / k g ) or daily a z a c o n a z o l e a d m i n i s t r a t i o n (0.1 or 0.3 m m o l / k g / d a y ) f o r 7 d a y s in male Fischer 3 4 4 rats. T h e in vitro effects o f a z a c o n a z o l e o n t h e a c c u m u l a t i o n o f organic ions b y renal cortical slices also were e x a m i n e d . A c u t e a z a c o n a z o l e a d m i n i s t r a t i o n (0.4 or 0.6 m m o l / k g , i.p.) p r o d u c e d a m a r k e d decrease in urine v o l u m e at 6 h. By 48 h urine v o l u m e was still decreased in t h e 0 . 6 - m m o l / k g g r o u p b u t n o t t h e 0 . 4 - m m o l / k g group. P r o t e i n u r i a (+ + ) and slight h e m a t u r i a were seen in t h e 0 . 6 - m m o l / k g g r o u p o n b o t h t r e a t m e n t days. A c c u m u l a t i o n o f p - a m i n o h i p p u r a t e (PAH) b y renal cortical slices was s t i m u l a t e d in b o t h a z a c o n a z o l e - t r e a t e d groups while t e t r a e t h y l a m m o n i u m {TEA) a c c u m u l a t i o n was n o t altered. No changes in b l o o d u r e a n i t r o g e n c o n c e n t r a t i o n , k i d n e y weight or renal m o r p h o l o g y were p r o d u c e d at 48 h p o s t i n j e c t i o n by e i t h e r a z a c o n a z o l e dose. Daily administ r a t i o n o f a z a c o n a z o l e (0.1 o r 0.3 m m o l / k g / d a y ) did n o t significantly alter a n y o f t h e renal p a r a m e t e r s studied. I n c u b a t i o n o f renal cortical slices with increasing c o n c e n t r a t i o n s o f a z a c o n a z o l e f r o m 10 -5 M to 10 -3 M caused a c o n t i n u e d r e d u c t i o n in T E A a c c u m u l a t i o n . PAH a c c u m u l a t i o n was decreased significantly following i n c u b a t i o n with a z a c o n a z o l e 10 -s or 10 -3 M. These results indicate t h a t a z a c o n a z o l e is capable of p r o d u c i n g a c u t e , reversible renal e f f e c t s at doses equal t o or less t h a n 0.6 m m o l / k g and altering organic ion t r a n s p o r t b o t h in vivo and in vitro. T h e lack o f m a r k e d renal effects
Abbreviations: BUN, blood urea nitrogen; H and E, hematoxylin and eosin; PAH, p-
aminohippurate; PAS, periodic acid Schiff; TEA, tetraethylammonium. 0300-483X/85/$03.30 © 1985 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
following exposure to azaconazole is favorable for the development of this c o m p o u n d as an agricultural fungicide.
Key words: Fungicides; Triazoles; hydrocarbons
Nephrotoxicity;
Rats;
Halogenated
INTRODUCTION The search for new, potent antifungal agents has resulted in the discovery of several promising fungicidal compounds. However, many of these compounds have been shown to be nephrotoxic in man or animal studies. For example, N-(3,5
N~N..PCl C~CI 0 /
"0--
I
I
AZACONAZOLE Fig. 1, Chemical structure of azaconazo|e (R 28644).
2
vivo and in vitro effects of azaconazole on renal function were examined. Renal tissue was examined for changes in morphology using light microscopy. METHODS Animals
Male Fischer 344 rats (200--300 g) were used in all experiments. Rats were purchased from Hilltop Lab Animals, Inc. (Scottdale, PA) and housed in rooms with controlled temperature (21--23°C), humidity and light (12 h light/12 h dark cycle). Rats were housed singly in stainless steel metabolism cages during the in vivo experiments. At all other times rats were housed in groups of not more than 6 rats per group in standard plastic animal cages. In vivo studies
Three days prior to their placement in metabolism cages, each rat had a blood sample taken from the tail for the determination of the basal blood urea nitrogen concentration (BUN, Sigma Kit No. 640). Rats were allowed free access to food (powdered Purina R o d e n t Chow) and water on the first day of individual housing. The next day (control day), food was removed around 0900 h and withheld for 6 h to assure collection of a urine specimen without food contamination. The 6-h urine specimens were analyzed qualitatively (Multistix ®, Ames Company) for the presence of protein, glucose, ketones and blood, and the results served as that day's urine content analysis. Body weight, food and water intake, and urine volume and osmolality were determined at 24-h intervals. Following the control day, rats (4 rats/group) in the acute studies received a single injection of vehicle only (sesame oil, 2.5 ml/kg, i.p.) or azaconazole (0.4, 0.6, or 1.0 mmol/kg, i.p.) at 0900 h and monitored as described above. At 48 h postinjection a blood sample was taken from the tail of each rat for BUN concentration determinations, and the rats were killed by cervical dislocation. Both kidneys were removed rapidly with the left kidney being used for the renal slice studies. The right kidney was weighed and examined for histological changes as described below. No animals died in the vehicle only groups or azaconazole 0.4- or 0.6-mmol/kg groups prior to sacrifice. However, all rats died in the azaconazole 1.0-mmol/kg group before 48 h, and their data are not included. In the subacute studies, rats (4 rats/group) received a daily i.p. injection of azaconazole (0.1 or 0.3 mmol/kg) or vehicle for 7 days. Urinary parameters, food and water intake, and b o d y weight were measured daily. BUN concentration, kidney weights, and organic ion accumulation by renal cortical slices were measured on day 7 as described in the acute studies. No animals died in any treatment or control group. Control rats were pair-fed for their appropriate treatment groups. This assured that any observed renal response was due to azaconazole treatment and not due to decreased food intake.
In vitro studies
In these studies rats were killed by cervical dislocation around 0900 h, and renal slices were prepared and processed as described below. Tissue slices were incubated with azaconazole for 30 min. [14C] PAH or [~4C] TEA were added, and the tissue incubation continued for 90 rain. Accumulation of PAH and TEA was determined as described below for the renal slice studies. Azaconazole solutions were prepared in 95% ethanol such that addition of 30/11 of the azaconazole solution would yield the desired final azaconazole concentration in the 3 ml of incubation medium. Each control sample had 30 t~l of 95% ethanol only added to the incubation medium. R e n a l slice studies
All rats were killed by cervical dislocation at the appropriate time. Kidneys were removed rapidly and placed in ice-cold Krebs-Ringer phosphate buffer (pH 7.4) containing 5.0 mM potassium and 1.0 mM calcium. Renal cortical slices were prepared freehand, and approximately 100 mg of tissue were incubated with shaking for 90 min in the same buffer at 25°C under a 100% oxygen atmosphere in a D u b n o f f metabolic shaker. Sodium lactate (10 -2 M) was used in some experiments. Following the incubation period, tissues were removed from the bathing media, blotted, weighed and homogenized in distilled water (4 ml) using a Tekmar tissumizer. Aliquots (1-ml) of the homogenates were placed in liquid scintillation counting vials and mixed with 9 ml of PCS counting solution. The incubation media were counted in a similar manner using 0.5-ml aliquots of media and 9.5 ml of PCS. Counting was performed using a Beckman LS-9000 liquid scintillation spectrometer with external standard quench correction. The uptake of p-['4C]aminohippurate (PAH) and ['4C]tetraethylammonium (TEA) was studied. For each compound, enough radioactive compound was added to yield about 0.02 #Ci of 14C/ml of incubation medium and sufficient unlabelled material to yield an initial concentration of 7 × 10 -S M for PAH and 10 -S M for TEA. Isotopes were obtained from New England Nuclear Corporation, Boston, MA. Accumulation of PAH and TEA by renal cortical slices was expressed as the slice to medium (S/M) ratio where S equaled the radioactivity (dpm)/g of tissue and M equaled the radioactivity (dpm)/ml of incubation medium. His t o logy
Following cervical dislocation, the right kidney was removed rapidly, weighed, cut in half lengthwise and placed in 10% neutral buffered formalin (3.7% formaldehyde in 75 mM sodium phosphate buffer, pH 7.0). Fixed tissues were embedded in paraffin, sectioned at 6--8 ~m and stained with hematoxylin and eosin (H and E) or periodic acid Schiff (PAS) reagent before histological examination. Sections of cortex, medulla and papilla were prepared and examined for histopathology using light microscopy (EPOI Lab I microscope). Tissues from treated animals were compared to tissues from pair-fed controls for the presence of chemically-induced changes in morphology.
Statistical analysis All data were analyzed using one-way analysis of variance and/ or Student's t-test. RESULTS
Acute in vivo experiments Azaconazole administration (0.4 or 0.6 mmol/kg) reduced food intake (P < 0.05) on bot h t r e a t m e n t days (Table I). To assure t hat any changes in renal f u n ction we observed were not due to decreased food intake, pair-fed c o n t r o l groups were used. The decrease in f o o d intake was accompanied by decreased water intake (Fig. 2). Water intake was n o t different between the azaconazole 0.4 m m o l / k g group and its pair-fed control group on any day, while the azaconazole 0.6-mmol/kg group drank significantly less water than their pair-fed c o n t r o l group on t r e a t m e n t day 1 but not on t r e a t m e n t day 2. b r i n e volume was markedly reduced at the 6-h measurement on both t r e a t m e n t days by azaconazole (0.4 or 0.6 mmol/kg). On t r e a t m e n t day 1 no urine was p r o d u c e d at 6 h in the 0.4-mmol/kg t r e a t m e n t group, and only 1 rat had voided urine, in the 0.6-mmol/kg t r e a t m e n t group. On day 2 urine volume was not significantly reduced in t he 0.4-mmol/kg group at 6 h but remained decreased in the 0.6-mmol/kg group. The 24-h urine volume was reduced b y azaconazole (0.4 or 0.6-mmol/kg) t r e a t m e n t on day 1. On day 2 the 24-h urine o u t p u t o f the 0.4-mmol/kg t r e a t m e n t group was not significantly reduced, but t he 0.6-mmol/kg t r e a t m e n t group still had significantly reduced urine o u t p u t . Urine volume was not altered in pair-fed control rats at th e 6-h or 24-h measurements on either t r e a t m e n t day. Urine osmolality was decreased in all groups on t r e a t m e n t day 1 when c o m p a r e d to appropriate p r e t r e a t m e n t values and in all groups except the 0.6-mmol/kg pair-fed c o n t r o l group on t r e a t m e n t day 2 (Fig. 2). On day 1, the decrease in urinary osmolality seen in t he 0.6-mmol/kg t r e a t m e n t group was significantly larger (P < 0.05) than the decrease in urinary osmolality TABLE I E F F E C T O F A Z A C O N A Z O L E ON F O O D I N T A K E a A z a c o n a z o l e dose (mmol/kg)
0.4 0.6
F o o d intake (g)b day0
day]
day2
18.14-1.5 18.04-1.2
4.5±0.6 c 1.44-0.6 c
11.64-0.4 c 9.54-2.1 c
aValues are m e a n s 4- S.E. for N = 4 rats. b F o o d intake was m e a s u r e d at 24-h intervals for t h e c o n t r o l day ( d a y 0) and at 24 h (day 1) a n d 48 h (day 2) p o s t i n j e c t i o n . c s i g n i f i c a n t l y d i f f e r e n t f r o m t h e day 0 value, P < 0.05.
5
c ~ 0-4mmol/kg ; control : v. 0-4mmol/kg ;treated 0-6m mol/kg ;control -" "- O'6mmol/kl ;treated
5-
15
E E • -,a
E
lO-
3
&.e,J Z 2i
~--
S
l-
DAYSAFTERTREATMENT
DAYSAFTERTREATMENT 30~0~ f
40EE 30 Z
*
~"
2000
~
]~
20-
10-
o
i
i
DAYSAFTERTREATMENT
DAYSAFTERTREATMENT
Fig. 2. E f f e c t o f a z a c o n a z o l e o n u r i n e v o l u m e , w a t e r i n t a k e a n d u r i n e o s m o l a l i t y . E a c h v a l u e is t h e m e a n a n d t h e v e r t i c a l line t h e s t a n d a r d e r r o r for N = 4. S o m e g r o u p s o f r a t s ( t r e a t e d ) w e r e a d m i n i s t e r e d a s i n g l e i.p. i n j e c t i o n o f a z a c o n a z o l e at t h e d o s e i n d i c a t e d while other groups (control) received vehicle only and were pair-fed for the appropriate t r e a t e d g r o u p . A n a s t e r i s k i n d i c a t e s P < 0 . 0 5 w h e n c o m p a r e d to t h e p r e t r e a t m e n t v a l u e for t h e p a r t i c u l a r g r o u p .
observed in t h e c o r r e s p o n d i n g pair-fed c o n t r o l g r o u p . T h e decreases observed in urinary o s m o l a l i t y in t r e a t e d groups o n day 2 were n o t d i f f e r e n t f r o m those seen in the a p p r o p r i a t e c o n t r o l groups. Urine c o n t e n t was n o t m a r k e d l y altered b y a z a c o n a z o l e . In t h e a z a c o n a z o l e 0 . 4 - m m o l / k g group, no urine was p r o d u c e d b y 6 h o n t r e a t m e n t d a y 1 so t h a t n o m e a s u r e m e n t s c o u l d be o b t a i n e d . On t r e a t m e n t d a y 2 o n l y h e m a t u r i a (trace) was n o t e d in this group. In t h e a z a c o n a z o l e 0 . 6 - m m o l / k g group, p r o t e i n u r i a ( + + ) and h e m a t u r i a (trace) were d e t e c t e d on b o t h t r e a t m e n t days. No urinary c o n t e n t changes were n o t e d in either pair-fed c o n t r o l group.
6
The BUN concentration in treated animals was not increased at 48 h by either dose of azaconazole. In addition, kidney weight at 48 h was not different between either treatment group and its pair-fed control. The accumulation of PAH by renal cortical slices was increased by azaconazole when compared to PAH accumulation by cortical slices from control rats, but TEA accumulation was not altered by azaconazole administration (Fig. 3). Basal PAH uptake was increased by azaconazole 0.4 mmol/kg, and lactate-stimulated PAH uptake was increased in both azaconazole treatment groups at 48 h postinjection. Increasing the dose of azaconazole to 1.0 mmol/kg resulted in the death of all treated rats by 30 h. Two animals died on treatment day 1 between 6 h and 24 h. The 2 animals surviving the first 24 h produced 0.6 and 2.0 ml/24 h. respectively. These remaining animals died on treatment day 2 before the 6-h urinary measurements. All rats were anuric at 6 h on treatment day 1, so that urine content measurements could not be performed, and eventually died in acute renal failure. Examination of renal tissue from azaconazole-treated rats at 48 h postinjection by light microscopy did not reveal any significant changes in renal morphology when compared to renal tissue from the appropriate pair-fed control group. Subacute in vivo experiments Since acute azaconazole administration (0.4 or 0.6 mmol/kg) appeared to produce only mild renal effects, it was felt that reducing the azaconazole dose and administering it daily (0.1 or 0.3 mmol/kg/day) for 7 days might [ " 0"4 m mol/kg'day ; control L~ 0-4 m mol/kg/day: treated
25
[ ] 0'6 m mol,'kg,,'day: control m o . G m mol/kg/day:treated 20 0
~-. 15 cC
10-
5
i
PAH
i
i
PAH + LACTATE
TEA
Fig. 3. E f f e c t o f a z a c o n a z o l e o n p - a m i n o h i p p u r a t e ( P A H ) a n d t e t r a e t h y l a m m o n i u m (TEA) a c c u m u l a t i o n by renal cortical slices. S o m e groups o f rats ( t r e a t e d ) were given a single i.p. i n j e c t i o n o f a z a c o n a z o l e at t h e dose indicated while o t h e r groups ( c o n t r o l ) received vehicle o n l y a n d were pair-fed for t h e a p p r o p r i a t e t r e a t e d group. K i d n e y s used in t h e s t u d y were o b t a i n e d at 48 h p o s t - i n j e c t i o n . T h e height o f each bar is t h e m e a n for N = 4. An asterisk indicates P < 0.05 w h e n c o m p a r e d to pair-fed controls.
7
20-
I.-,-
-" --
-" --
PAH PAH + LACTATE
-"
--
TEA
IS-
()
1026M 1025M 1024M lO-'3M
AZACONAZOLECONCENTRATION Fig. 4. In vitro effects o f a z a c o n a z o l e o n p - a m i n o h i p p u r a t e (PAH) and t e t r a e t h y l a m m o n i u m ( T E A ) a c c u m u l a t i o n b y renal cortical slices. Each value r e p r e s e n t s the m e a n and t h e vertical line the s t a n d a r d error for N = 4 - 6 e x p e r i m e n t s . A n asterisk indicates P < 0.05 w h e n c o m p a r e d to c o n t r o l e x p e r i m e n t s .
reveal wh ethe r or not exposure to azaconazole produces n e p h r o t o x i c i t y in rats. Although f o o d intake was slightly but significantly reduced for 2 days {data not shown) in the azaconazole 0.1-mmol/kg/day group, the renal parameters studied were not altered. Likewise, azaconazole 0.3 m m o l / k g / d a y administration did n o t consistently alter renal function. BUN concentration, kidney weight and organic ion accumulation by renal cortical slices were not different among t he azaconazole t r e a t e d (0.1 or 0.3 m m o l / k g / d a y ) and their pair-fed co n t r ol groups. Renal m o r p h o l o g y was not altered in either treatmerit group when c o m p a r e d to c ont r ol animals. In vitro experiments Renal cortical slices incubated with azaconazole at various concentrations exhibited different tendencies for accumulating the organic anion PAH and the organic cation T E A (Fig. 4). Basal and lactate-stimulated PAH and T E A accumulations steadily declined as the azaconazole c o n c e n t r a t i o n in the incubation media increased. All 3 transport parameters were decreased significantly in t he presence of 10 -s M azaconazole, but basal and lactatestimulated PAH accumulation increased to cont rol levels in bathing media containing 10-4 M azaconazole. With a c o n c e n t r a t i o n o f 10 -3 M azaconazole in the media bot h basal and lactate-stimulated PAH accumulations were again significantly decreased. DISCUSSION
The results of this study d e m o n s t r a t e t hat azaconazole can p r o d u c e reversible effects on renal parameters in rats following acute administration of nonlethal doses and alter organic ion accumulation by renal cortical slices following in vivo or in vitro exposure.
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The production of oliguria or anuria in rats treated acutely with azaconazole was rapid in onset and reversible at doses of 0.6 mmol/kg or lower. Similar reversible renal dysfunction has been reported following administration of therapeutically useful antifungal agents [12--15] or exposure to agricultural fungicides [16]. However, renal dysfunction may contribute to death at lethal azaconazole concentrations, since all rats in the 1.0mmol/kg dose group died in acute renal failure. Although urinary osmolality decreased following azaconazole administration, it is unlikely that this is a primary effect of azaconazole on the kidney. Both pair-fed control groups also excreted a more dilute urine on treatment day 1, and the control group for the 0.4-mmol/kg treatment group exhibited decreased urinary osmolality on treatment day 2. These observations emphasize the importance of using pair-fed control groups for studies involving compounds which markedly depress food intake. Organic ion transport was alerted by azaconazole after in vivo or in vitro exposure. However, the extent of organic ion accumulation varied depending upon the m e t h o d of exposure to azaconazole, azaconazole dose or concentration, and the organic ion studied. In the in vivo experiments, PAH accumulation was stimulated at 48 h postinjection, while TEA accumulation was not altered. Similar increases in PAH uptake without altering organic cation accumulation have been observed following administration of gentamicin [17], pentobarbital [18], potassium dichromate [19,20] or trish y d r o x y m e t h y l a m i n o m e t h a n e [21]. In addition, a number of metabolic intermediates of the glycolytic and Krebs pathways stimulate both PAH and TEA transport with PAH transport being enhanced to a much greater extent [22--24]. The reasons for this difference in the accumulation of organic anions and cations is not known with certainty. These effects might be related to an interaction of the test c o m p o u n d with specific sites in the proximal tubules [19] or to a decreased efflux of accumulated organic ion [251. Pretreatment of renal cortical slices in vitro with azaconazole resulted in a decline in PAH (basal and lactate-stimulated) and TEA accumulation. Return of PAH accumulation to control S/M ratios in the presence of 10 -~ M azaconazole probably reflects the variability of the small sample size. The marked decrease in both organic anion and cation accumulation at 10 -3 M azaconazole suggests a generalized toxic effect on all segments of the proximal tubules. Although azaconazole produces acute renal effects in rats, other experimental agricultural fungicides have been shown to produce more severe renal dysfunction. For example, a single 0.4 mmol/kg, i.p. injection of N-(3,5-dichlorophenyl)succinimide to Sprague--Dawley rats produces marked diuresis, proteinuria, glucosuria and hematuria, elevates BUN concentration, decreases accumulation of PAH and TEA by renal cortical slices and produces acute tubular necrosis [4]. Similar results are obtained in Fischer 344 rats (unpublished results). Administration of azaconazole (0.4 mmol/kg, i.p.) produced oliguria but did not elevate BUN, decrease PAH or TEA accumu-
lation or alter renal m or phol ogy. Thus, azaconazole does not produce the severity o f renal effects seen with some experimental fungicides. In summary, azaconazole is an experimental agricultural fungicide capable of producing acute, reversible renal effects at doses equal to or less than 0.6 mmol/kg. Organic ion transport is altered bot h in vivo and in vitro, but the mechanisms producing these changes are not clear. The lack of marked renal d y s f u n c t i o n following subacute exposure or altered renal m o r p h o l o g y after acute or subacute exposure suggests that any long lasting effects on renal function p r oduc e d by azaconazole are probably minor. Therefore, azaconazole might prove to be safer as an agricultural fungicide than other compounds currently being developed, since unlike dimetachlone and some ot her experimental fungicides, azaconazole does not appear to be n e p h r o t o x i c following acute or subacute exposure in rats. ACKNOWLEDGEMENTS
Th e authors would like to thank Janssen Pharmaceutica, Inc. for their generous gift of azaconazole. T he authors also would like to thank E. Patricia Lahoda and Hermandra Shah for their excellent technical assistance. REFERENCES I A. Fujinami, T. Ozaki and S. Yamamoto, Studies on biological activity of cyclic imide compounds. Part I. Antimierobial activity of 3-phenyloxazolidine-2,4-diones and related compounds. Agric. Biol. Chem., 35 (1971) 1707. 2 A. Fujinami, T. Ozaki, K. Nodera and K. Tanaka, Studies on biological activity of cyclic imide compounds. Part II. Antimicrobial activity of 1-phenylpyrrolidine-2,5diones and related compounds. Agric. Biol. Chem., 36 (1972) 318. 3 A. Fujinami, N. Tottri, T. Kato and N. Kameda, Antimierobial activity of 3-phenyloxazolidine-2,4-diones and related compounds. Agrie. Biol. Chem., 36 (1972) 1623. 4 G.O. Rankin, Nephrotoxicity following acute administration of N-(3,5-diehlorophenyl)suceinimide in rats. Toxicology, 23 (1982) 21. 5 G.O. Rankin, K. Cressey-Veneziano and P.I. Brown, Onset of and recovery from acute N-(3,5-diehlorophenyl)succinimide-induced nephrotoxieity in Sprague--Dawley rats. Toxicology, 30 (1984) 205. 6 S. Sugihara, Y. Shinohara, Y. Miyata, K. Inoue and N. Ito, Pathologic analysis of chemical nephritis in rats induced by N-(3,5-dichlorophenyl) succinimide. Lab. Invest., 33 (1975) 219. 7 E.F. Godefroi, d. Heeres, J. Van Cutsem and P.A.J. Janssen, The preparation and antimyeotic properties of derivatives of 1-phenethylimidazole. J. Med. Chem., 12 (1969) 784. 8 J. Heeres, L.J.J. Baekx and J. Van Cutsem, Synthesis and antimycotic properties of 1-(2-alkyl-2-phenylethyl)-lH-imidazoles. J. Med. Chem., 19 (1976) 1148. 9 J. Van Cutsem and D. Thienpont, Miconazole, a broad spectrum antimyeotic agent with antibacterial activity. Chemotherapy (Basel), 17 (1972) 392. 10 D.H. Bartlett and N . E Ballard, The effectiveness of Gauzatine and Imazalil as seed treatment fungicides in barley. Proc. Br. Insectic. Fungic. Conf., 8th, 1 (1975) 205. 11 J. Van Gestel, J. Heeres, M. danssen and G. Van Reet, Synthesis and screening of a new group of fungicides: 1-(2-Phenyl-l,3-dioxolan-2-ylmethyl)-l,2,4-triazoles. Pestle. Sei., 11 (1980) 95.
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