See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/13976493 Cytochrome P450-Mediated Metabolism and Nephrotoxicity of N-(3,5- Dichlorophenyl)succinimide in Fischer 344 Rats Article  in  Fundamental and Applied Toxicology · July 1997 DOI: 10.1093/toxsci/37.2.117 · Source: PubMed CITATIONS READS 14 39 3 authors, including: Alexander Kwadwo Nyarko Ginny L Weibel University of Ghana The Children's Hospital of Philadelphia 141 PUBLICATIONS   1,193 CITATIONS    30 PUBLICATIONS   1,330 CITATIONS    SEE PROFILE SEE PROFILE Some of the authors of this publication are also working on these related projects: Mechanism underlying the spasmolytic and anti-inflammatory effects of the fruit juice of Morinda citrifolia View project Bioassay guided isolation and structure elucidation of antimycobacterial compounds View project All content following this page was uploaded by Alexander Kwadwo Nyarko on 28 May 2014. The user has requested enhancement of the downloaded file. FUNDAMENTAL AND APPLIED TOXICOLOGY 37, 1 17 -124 (1997) ARTICLE NO. FA972321 Cytochrome P450-Mediated Metabolism and Nephrotoxicity of /V-(3,5- Dichlorophenyl)succinimide in Fischer 344 Rats Alexander K. Nyarko,* Ginny L. Kellner-Weibel.f and Peter J. Harvison:}:1 *Chemical Pathology Unit, Noguchi Memorial Institute, University of Ghana, Legon-Accra, Ghana; and Departments of ^Chemistry and tPharmacology and Toxicology, Philadelphia College of Pharmacy and Science, 600 South Forty-third Street, Philadelphia, Pennsylvania 19104-4495 Received December 3, 1996; accepted April 21, 1997 icity studies indicated that NDPS induces a nephrotic syn- Cytochrome P450-Mediated Metabolism and Nephrotoxicity of drome in rats that is similar to human interstitial nephritis A/-(3,5-Dichlorophenyl)succinimide in Rats. Nyarko, A. K., Kell- ner-Weibel, G. L., and Harvison, P. J. (1997). (Sugihara et al, 1975; Barrett et al, 1983). In acute toxic- Fundam. Appl. Tox- icol. 37, 117-124. ity studies, NDPS produced diuresis, proteinuria, glucos- uria, and elevated blood urea nitrogen; alterations in renal The agricultural fungicide N-(3,5-dichlorophenyl)succinimide organic ion uptake and increased kidney weights were (NDPS) is nephrotoxic in rats. Previous studies have suggested also reported (Rankin, 1982; Rankin et al., 1984, 1985; that oxidative hepatic biotransformation is required for the induc- Kellner-Weibel et al, 1995). Sugihara et al. (1975) sug- tion of kidney damage. The experiments described in this paper gested that NDPS is a potentially useful model compound were designed to further investigate the relationship between for studying chemically induced interstitial nephritis. In NDPS metabolism and nephrotoxicity using various modulators of cytochrome P450 activity. Male Fischer 344 rats were pretreated fact, the succinimide ring, which is important in NDPS with the P450 inducers Aroclor 1254 (ARO), isoniazid (INH), 3- nephrotoxicity (see below), is also found in many other methylcholanthrene (3-MC), and phenobarbital (PB), or the P450 compounds (including drugs, agricultural agents, and in- inhibitor 1-aminobenzotriazole (ABT). Control animals received dustrial chemicals) to which humans and other species vehicle only. NDPS metabolism was investigated using hepato- can be exposed. cytes isolated from the various treatment groups. Separate experi- Previous studies (Ohkawa et al., 1974; Griffin and Harvi- ments were also conducted to evaluate the effects of these pre- son, 1990) demonstrated that NDPS undergoes in vivo metab- treatments on NDPS-induced nephrotoxicity in rats. PB and ARO olism in rats (Fig. 1) to Af-(3,5-dichlorophenyl)succinamic enhanced formation of the known nephrotoxic NDPS metabolites, N-(3,5-dichlorophenyl)-2-hydroxysuccinimide, N-(3,5-dichloro- acid (NDPSA), A^-(3,5-dichlorophenyl)-2-hydroxysuccinamic phenyl>2-hydroxysuccinamic acid, and iV-(3,5-dichlorophenyl)-3- acid (2-NDHSA), /V-(3,5-dichlorophenyl)-3-hydroxysucci- hydroxysuccinamic acid, by the hepatocytes. In contrast, ABT namic acid (3-NDHSA), and N-(3,5-dichlorophenyi)malo- inhibited formation of the nephrotoxic metabolites, whereas INH namic acid (DMA). NDPSA, 2-NDHSA, 3-NDHSA, ^-(3,5- and 3-MC did not alter NDPS biotransformation. NDPS-induced dichlorophenyl)-2-hydroxysuccinimide (NDHS), and /V-(3,5- renal damage was potentiated by pretreating the rats with PB or dichloro-4-hydroxyphenyl)succinamic acid (NDHPSA) were ARO and was attenuated by ABT. Compared with control ani- detected when NDPS was incubated with isolated rat hepato- mals, toxicity was unaffected by INH or 3-MC pretreatments. cytes or rat liver homogenates (Nyarko and Harvison, 1995; Thus, there was a correlation between pretreatments that induce Griffin et al., 1996). No oxidative metabolites were found in P450-mediated NDPS metabolism and the effects that these com- incubations of NDPS with isolated rat kidney cells (Henesey pounds have on NDPS-induced nephrotoxicity. The data indicate and Harvison, 1995). that specific P450 isozymes metabolize NDPS to its hydroxylated products and suggest that these metabolites mediate the nephro- The actual mechanism leading to NDPS-induced nephro- toxicity induced by NDPS. © 1997 Sodtty of To toxicity is not known, but metabolic activation by cyto- chrome P450 is strongly implicated. For example, deuterium labeling of the succinimide ring attenuated the renal damage associated with NDPS (Rankin et al, 1986). In addition, iV-(3,5-Dichlorophenyl)succinimide (NDPS, Fig. 1) was modulation of NDPS toxicity by induction or inhibition of originally synthesized in the early 1970s as an agricultural microsomal metabolism suggested that hepatic cytochrome fungicide (Fujinami et al, 1972). Subsequent chronic tox- P450 was responsible for metabolic activation of this com- pound (Rankin etal, 1987). Finally, several hepatic metabo- ' To whom correspondence should be addressed. Fax: 215-895-1161. E- lites (NDHS, 2-NDHSA, and 3-NDHSA) were found to be mail: p.harvis@pcps.edu. more nephrotoxic than NDPS itself (Rankin et al, 1988, 117 0272-0590/97 $25.00 Copyright © 1997 by the Society of Toxicology. All rights of reproduction in any form reserved. Downloaded from toxsci.oxfordjournals.org by guest on July 13, 2011 18 NYARKO, KELLNER-WEIBEL, AND HARVISON NDPS NDHS COjH NDPSA 3-NDHSA 2-NDHSA FIG. 1. of the product was immediately measured on a Hitachi F-3010 spectroflu- ARO SKF 525A 4 7.3 ± 1.2 62.1 ± 2 .3 ' ' ' r orometer (excitation wavelength 395 nm, emission wavelength 522 nm) 3-MC None 4 9.1 ± 2.6 59.2 ± 2.2 C against zero time and reagent blanks. Product formation was quantitated I4 6 by comparing the fluorescence of the samples with standards generated by ° [ C]NDPS (2.0 mM) and hepatocytes (5 X 10 cells/ml) were incubated quinine sulfate prepared in 0.5 M HjSC^ (Nebert and Gelboin, 1968). in Krebs-Henseleit buffer (pH 7.4, 37°C) for 3 hr. Some incubations con- tained SKF 525A (50 / / M ) . Hydroxylation of p-nitrophenol (PNP) to 4-nitrocatechol was determined * Results are expressed as means ± SE. "Nephrotoxic metabolites" rep- by the method of Reinke and Moyer (1985). Each incubation contained ca. resents the sum of 2-NDHSA, 3-NDHSA, and NDHS. "Other metabolites" 1 mg microsomal protein, 0.4 mg NADPH, and 5.0 mM MgCl2 in a total is the sum of NDPSA and NDHPSA. volume of 1 ml Tris buffer (pH 7.4, 37°C). The reactions were initiated by c Significantly different from the saline (ABT, INH, PB) or corn oil- the addition of 100 nmol PNP and were terminated after 10 min by the pretreated (ARO, 3-MC) groups (p < 0.05). addition of 0.5 ml 0.6 N HC1O4 to each incubation. Precipitated proteins ' Significantly different from the corresponding group without SKF 525A were removed by centrifugation at 2500 rpm. Aliquots (1 ml) of the superna- (p < 0.05). tants were then mixed with 0.1 ml of 10 M NaOH. The absorbance of each sample was read at 546 nm (Hitachi U-3110 spectrophotometer) and the amount of 4-nitrocatechol formed was calculated using a molar absorptivity of 10.28 mM~'cnr' (Reinke and Moyer, 1985). RESULTS In vivo nephrotoxicity. Toxicity studies were conducted as previously described (Kellner-Weibel el al., 1995). Rats were transferred into plastic Production of the nephrotoxic metabolites (2-NDHSA, 3- metabolism cages for acclimatization prior to the in vivo toxicity studies. NDHSA, and NDHS) was enhanced 6-fold in the PB-in- Blood samples were collected by nicking the tails for determination of duced cells when compared with the uninduced cells (Table initial blood urea nitrogen (BUN) levels. The animals were then pretreated 1). In contrast, formation of the other metabolites (NDPSA as described above. On Day 0 (control day) of the experiment, food and and NDHPSA) was reduced, although not significantly (p water were removed for a 6-hr period to ensure collection of contaminant- > 0.05), by PB induction. Preincubation of PB-induced cells free urine. This urine was semiquantitatively analyzed (N-Multistix SG, Miles, Inc., Elkhart, IN) for the presence of protein, glucose, blood, and with the P450 inhibitor SKF 525A inhibited the production ketones. Food and water were then returned to the rats. Following the of the nephrotoxic metabolites. Hepatocytes isolated from control day, the rats were injected with NDPS (0.2 or 0.4 mmol/kg, ip in ARO-pretreated rats produced a 3.5-fold greater amount of com oil) or corn oil only (2 ml/kg). After dosing, the rats were returned to the toxic metabolites than the uninduced cells (Table 1). the metabolism cages for an additional 48 hr. Urine contents (analyzed as Formation of the other metabolites was significantly reduced above), total urine volume, body weight, and food and water consumption in the ARO-induced cells. In addition, ARO-induced cells were monitored each day of the experiment. A final blood sample was taken by cardiac puncture under methoxyflurane anesthesia 48 hr postdosing. converted NDPS to a previously undetected metabolite (7.07 6 While still anesthetized the animals were sacrificed by cervical dislocation. ± 1.15 nmol/10 cells/hr) which eluted immediately after The right kidney was then removed and weighed. BUN levels were deter- the solvent front (retention time = 1.8 min). SKF 525A mined using a commercially available kit (Kit 640-A, Sigma Chemical Co., reduced the production of the nephrotoxic metabolites (Table St. l-nuis, MO). 1) and the unknown metabolite (not shown) by hepatocytes Statistics. The data were analyzed by Student's t test, one way ANOVA, isolated from ARO-pretreated rats. In contrast to the results or the corresponding nonparametric tests. Where significance was detected obtained with PB or ARO, conversion of NDPS to the toxic in the ANOVA, the Student-Newman-Keuls, Dtinnett, or Dunn test was metabolites was not enhanced by the 3-MC or INH pretreat- used to isolate differences among the groups. A 5% level of significance ment (Table 1). Hepatocytes isolated from rats pretreated was used for all statistical analyses. Results are presented as means ± standard errors of the mean (SE) with N = 4, unless otherwise indicated. with ABT produced significantly less of the nephrotoxic A larger group size was used in the in vivo toxicity studies due to the metabolites than control cells (Table 1). variability in response to NDPS. When expressed as a percentage, the nephrotoxic metabo- Downloaded from toxsci.oxfordjournals.org by guest on July 13, 2011 120 NYARKO, KELLNER-WBD3EL, AND HARV1SON lites accounted for approximately 50-65% of the total radio- activity (metabolites + unreacted substrate) in the incuba- tions with the PB- or ARO-induced cells. In comparison, the toxic metabolites accounted for only 10-15% of the total using hepatocytes that were isolated from INH- or 3-MC- pretreated rats. ABT pretreatment reduced formation of the nephrotoxic metabolites to only 1% of the total. To verify that enzyme induction had actually occurred with PB, INH, ARO, and 3-MC, the cytochrome P450 con- tent of the liver cells was determined by CO difference spec- tra. As shown in Fig. 2, hepatocyte P450 levels were in- creased 2.5- to 4-fold by the various inducers when com- pared with cells isolated from animals that were pretreated with saline or corn oil (0.42-0.46 nmol P450/106 cells). To prove that induction of specific P450 isozymes had occurred with 3-MC and INH, experiments were conducted using liver microsomes prepared from rats that were pretreated with these two compounds. BP and PNP were used as substrates of the 3-MC- and INH-inducible isozymes, respectively. Mi- crosomal P450 levels were also assayed. As shown in Fig. 3A, the P450 content of microsomes isolated from saline- or corn oil-pretreated (control) rats was 0.80 nmol/mg pro- tein. This was increased ca. 220% by 3-MC induction. BP hydroxylase activity (3.25-4.10 mg product/min/mg protein in control microsomes) was elevated ca. 2-fold by 3-MC pretreatment, but was unaltered by INH induction (Fig. 3B). Hydroxylation of PNP in the uninduced liver microsomes (0.98 nmol product/min/mg protein) was enhanced approxi- mately 1.5-fold by pretreating rats with INH (Fig. 3C). i- 0 Saline INH Corn 3-MC oil PRETREATMENT FIG. 3. Effect of cytochrome P450 modulation on (A) microsomal P450 content, (B) benzo[a]pyrene (BP) hydroxylation activity, and (C) p-ni- trophenol (PNP) hydroxylation. P450 levels were determined by carbon monoxide difference spectra. Incubations contained BP or PNP (100 nmol each), bovine serum albumin (0.5 mg, BP assay only), NADPH (0.4 mg), MgCl2 (5 mM), and microsomes (ca. 1 mg) in 1 ml Tris buffer (0.05 mM, pH 7.4, 37°C). Product formation was quantitated fluorometrically (BP hydroxylation) or spectrophotometrically (PNP hydroxylation). Results are expressed as means ± SE (N = 3). Asterisks indicate values that are signifi- cantly different (p < 0.05) from saline controls. Daggers indicate values that are significandy different (p < 0.05) from com oil controls. Saline PB INH Corn 3-MC ARO oil The effects of the various pretreatments on NDPS-induced PRETREATMENT nephrotoxicity was evaluated in male Fischer 344 rats. Com- pared with animals (controls) that received corn oil instead FIG. 2. Effect of cytochrome P450 modulation on hepatocyte P450 of NDPS, BUN levels were increased at least 6-fold in ARO- content. P450 levels were determined by carbon monoxide difference spec- or PB-pretreated rats that received a nontoxic dose (0.2 tra. Results are expressed as means ± SE (N = 4). Asterisks indicate values that are significantly different (p < 0.05) from saline controls. Daggers mmol/kg) of NDPS or nonpretreated rats that received a indicate values that are significantly different (p < 0.05) from com oil toxic dose (0.4 mmol/kg) of NDPS (Table 2). The elevations controls. in BUN for these three treatment groups were also greater Downloaded from toxsci.oxfordjournals.org by guest on July 13, 2011 NDPS METABOLISM AND NEPHROTOXICITY 121 TABLE 2 Effect of Cytochrome P450 Modulation on N-(3,5-Dichlorophenyl)succinimide (NDPS)-Induced Changes in Blood Urea Nitrogen (BUN) Levels and Kidney Weights in Male Fischer 344 Rats BUN concentration (mg/dl)* NDPS dose Kidney weight* Pre treatment (mmol/kg) N Day 0 Day 2 (g/100 g body wt) None 0.0 4 20.1 ± 1.3 19.9 ± 0.7 0.39 ± 0.01 ARO 0.0 4 18.7 ± 0.2 21.9 ± 0.5' 0.39 ± 0.01 PB 0.0 4 18.7 ± 0.6 24.4 ± O.^ 0.36 ± 0.004 None 0.2 8 21.6 ± 0.9 49.7 ± 22.0* 0.45 ± 0.03 ARO 0.2 7 21.6 ± 1.2 124.6 ± 34.4'J 0.51 ± 0.05 INH 0.2 4 19.5 ± 0.6 25.3 ± 1.6' 0.36 ± 0.01 3-MC 0.2 7 19.0 ± 0.8 44.9 ± 22.6' 0.42 ± 0.04 PB 0.2 7 21.3 ± 0.9 149.8 ± 35.4°* 0.54 ± 0.04 None 0.4 4 21.7 ± 1.3 155.2 ± lO.O'-' 0.58 ± 0.03 ABT 0.4 4 17.5 ± 0.4 16.5 ± 0.7 0.42 ± 0.004 ° NDPS was administered at two doses: 0.2 mmol/kg (nontoxic) and 0.4 mmol/kg (nephrotoxic). Control rats (0.0 mmol/kg NDPS) received corn oil only (2 ml/kg). * Results are expressed as means ± SE. e Significantly different from the Day 0 value within a group (p < 0.05). * Significantly different from the nonpretreated control group (p < 0.05). than the values obtained from the same animals before NDPS treatment groups continued to lose weight on Day 2 of the was administered (Day 0 values). Relative to corn oil con- experiment. Body weights were not adversely affected in trols, 3-MC or INH induction had no effect on urea nitrogen any other treatment group. Compared with corn oil controls, levels in rats that received 0.2 mmol/kg NDPS (Table 2). food intake on Days 1 or 2 was reduced in the 0.4 mmol/ Pretreatment with ABT reversed the increase in BUN that kg NPDS, ARO + 0.2 mmol/kg NDPS, and PB + 0.2 mmol/ was associated with the nephrotoxic dose of NDPS. The kg NDPS treatment groups (data not shown). In addition, ARO or PB pretreatments had no effect on BUN levels in food intake in these groups after dosing with NDPS was rats that received corn oil instead of NDPS. As shown in lower than on Day 0. Rats in the 0.2 mmol/kg NDPS and Table 2, kidney weights were elevated in ARO- or PB- pretreated animals that received 0.2 mmol/kg NDPS or non- pretreated rats that received 0.4 mmol/kg NDPS, although these changes were not statistically significant (p > 0.05). TABLE 3 Effect of Cytochrome P450 Modulation on N-(3,5-Dichloro- Kidney weights were normal in the other treatment groups, phenyl)succinimide (NDPS)-Induced Changes in Urine Volume including rats that received ABT prior to a nephrotoxic dose in Male Fischer 344 Rats of NDPS (Table 2). Compared with Day 0, diuresis was present on Day 1 in Urine volume (ml)' rats that were pretreated with ARO, 3-MC, or PB before NDPS dose dosing with NDPS (0.2 mmol/kg) or in animals that received Pretreatment (mmol/kg) N Day 0 Day 1 Day 2 a toxic dose of NDPS (Table 3). Diuresis was completely None 0.0 4 12.3 ± 1.8 12.9 :fl.O 14.8 ± 1.3 prevented by pretreating rats with ABT 1 hr prior to adminis- ARO 0.0 4 10.5 ± 0.9 11.0 :t 0.4 11.5 ± 0.9 tration of NDPS (0.4 mmol/kg). Urine output was not altered PB 0.0 4 16.0 ± 1.4 13.5 :t 0.3 13.8 ± 1.7 by the other treatments and returned to normal levels in the None 0.2 8 13.3 ± 0.9 22.3 :t 3.4 14.6 ± 3.2 affected groups by Day 2. ARO 0.2 7 14.8 ± 1.3 28.6 :t 1.6' 17.7 ± 2.4 INH 0.2 4 13.8 ± 0.5 15.0 :t 2.0 15.5 ± 1.5 Twenty-four hours after a nephrotoxic dose of NDPS (0.4 3-MC 0.2 7 9.4 ± 0.7 22.0 jt 3.7' 14.9 ± 3.0 mmol/kg) was administered, there was a decrease (ca. 12%) PB 0.2 7 15.6 ± 1.0 28.1 jt 2.4' 20.7 ± 2.1 in body weight compared with corn oil controls (Table 4). None 0.4 4 11.3 ± 1.5 24.5 it i.r 15.8 ± 3.3 This decrease persisted into Day 2 and was prevented by ABT 0.4 4 11.0 ± 0.4 9.3 :t 0.9 12.8 ± 1.8 pretreating the animals with ABT prior to dosing with NDPS. " NDPS was administered at two doses: 0.2 mmol/kg (nontoxic) and 0.4 Relative to Day 0, decreases in body weight on Day 1 were mmol/kg (nephrotoxic). Control rats received com oil only (2 ml/kg). found in rats that received 0.2 mmol/kg NDPS (naive, ARO- * Results are expressed as means ± SE. or PB-pretreated) or 0.4 mmol/kg NDPS. Animals in these ' Significantly different from the Day 0 value within a group (p < 0.05). Downloaded from toxsci.oxfordjournals.org by guest on July 13, 2011 122 NYARKO, KELLNER-WEIBEL, AND HARVISON TABLE 4 the effect that these pretreatments have on in vitro and in Effect of Cytochrome P450 Modulation on N-(3,5-Dichloro- vivo NDPS biotransformation. We were interested in de- phenyl)succinimide (NDPS)-Induced Changes in Body Weight termining if modulation of NDPS toxicity would correlate in Male Fischer 344 Rats with changes in metabolism. Male Fischer 344 rats were therefore pretreated with a P450 inhibitor (ABT) or com- Body weight (g)* pounds (ARO, INH, 3-MC and PB) that induce different NDPS dose? Pretreatment (mmol/kg) N Day 0 Day 1 Day 2 P450 isozymes. The animals were then used to evaluate NDPS toxicity and metabolism. NDPS was administered to None 0.0 4 186.1 ± 3.8 184.7 ±4.8 1883 :t 5.4 rats at two doses: 0.2 mmol/kg (nontoxic) and 0.4 mmol/kg ARO 0.0 4 176.7 1t 3.3 180.6 ± 3.0 184.4 :t 3.6 (toxic). These doses were chosen based on reports in the PB 0.0 4 204.9 2t 5.8 202.5 ± 5.8 205.1 :t5.6 t3 .4 ' literature (Rankin, 1982; Rankin etal., 1984, 1985; Kellner-None 0.2 8 207.1 ;t 2.3 194.6 ± \.T 197.9 : ARO 0.2 7 215.1 =t 2.7 195.5 ± 4.T 190.4:t6 .4 ' Weibel et al., 1995). Metabolism experiments were con- INH 0.2 4 195.4 :t 5.1 195.8 ± 53 197.0 :t5.2 ducted using isolated rat liver cells as this system produces 3-MC 0.2 7 195.3 ;t 3.6 184.8 ± 4.5 188.0 ± 5.9 most of the known, in vivo NDPS metabolites (Nyarko and PB 0.2 7 208.9 ;t 3.2 184.6 ± 3.8* 1763 :t5.5 c Harvison, 1995). Prior studies have shown that the biotrans- None 0.4 4 179.4 2t 2.7 157.5 ± 22^ 146.6 ± 2.0^ formation of other compounds, such as acetaminophen (Mol- ABT 0.4 4 174.2 2t \5 170.8 ± 22 1776 ± 2.5 deus, 1978) and nicotine (Kyerematen et al., 1990), can be ° NDPS was administered at two doses: 0.2 mmol/kg (nontoxic) and 0.4 modified in rat hepatocytes by enzyme induction. mmol/kg (nephrotoxic). Control rats received com oil only (2 ml/kg). The freshly isolated rat liver cells converted NDPS to * Results are expressed as means ± SE. c Significantly different from the Day 0 value within a group (p < 0.05). NDPSA, 2-NDHSA, 3-NDHSA, NDHS, and NDHPSA. Me- ** Significantly different from the nonpretreated control group (p < 0.05). tabolite production by hepatocytes isolated from saline- or corn oil-pretreated rats (indicative of constitutive P450 activ- ABT + 0.4 mmol/kg NDPS groups also ate less in the first 24 ity) was quantitatively similar to our prior results (Nyarko hr after dosing; however, food consumption in these animals and Harvison, 1995). Pretreatment of rats with ARO, INH, returned to normal on Day 2. Water consumption on Days 3-MC, or PB significantly increased the cytochrome P450 1 and 2 was not significantly different from that of corn oil content of the hepatocytes, indicating that enzyme induction controls in any treatment group during the course of the was successful. The P450 levels in our control and PB- and experiment (data not shown); however, rats in the 0.4 mmol/ 3-MC-induced liver cell preparations were comparable to kg NDPS and PB + 0.2 mmol/kg NDPS groups drank sig- those reported by Moldeus et al. (1973). nificantly less water for the first 24 hr after dosing. Water In agreement with Rankin et al. (1987), we found that PB intake in these animals returned to normal on Day 2. pretreatment potentiated NDPS (0.2 mmol/kg) nephrotoxic- Semiquantitative urinalysis showed that proteinuria ity in rats. PB is an inducer of the P450 2B1/2B2 isozymes (>100 mg/dl) occurred within 6 hr in rats that received in rat liver (Guengerich et al., 1982), but has no effect on 0.2 mmol/kg NDPS (nonpretreated, ARO-, 3-MC-, and PB- rat renal P450 (Tarloff et al., 1990). Kidney damage in the induced) or 0.4 mmol/kg NDPS (data not shown). Urine PB-pretreated rats was evident as increased diuresis, eleva- protein levels remained elevated in all of these groups on tions in blood urea nitrogen levels and kidney weights, pro- Day 2. ABT pretreatment totally prevented the proteinuria teinuria and glucosuria, and decreased body weights and associated with the nephrotoxic dose of NDPS. Urine protein food intake. We previously reported that these changes cor- levels were essentially normal in all other animals. Glucosu- related with histological damage in rats that received a neph- ria (=*100 mg/dl) occurred in the 0.4 mmol/kg NDPS and rotoxic dose (0.4 mmol/kg) of NDPS (Kellner-Weibel et al., 0.2 mmol/kg NDPS (ARO- and PB-induced) animals 6 hr 1995). ARO, which is an inducer of the P450 1A and 2B after administration of the compound (data not shown). isozyme families (Guengerich et al., 1982), also enhanced Urine glucose levels remained elevated in the PB + 0.2 NDPS nephrotoxicity. The variability in BUN values ob- mmol/kg NDPS and 0.4 mmol/kg NDPS treatment groups tained from rats that received 0.2 mmol/kg NDPS following on Day 2. The glucosuria associated with 0.4 mmol/kg pretreatment with PB or ARO (Table 1) may be due to the NDPS was prevented by ABT pretreatment. All other treat- "steep" dose-response curve exhibited by NDPS in Fischer ment groups had normal urine glucose levels. There was no 344 rats (Rankin et al., 1985). Increasing the number of evidence of ketonuria or hematuria in any animals through- animals in these experiments failed to reduce this variability. out the experiment (data not shown). Renal damage was not observed in PB- or ARO-pretreated rats that received com oil instead of NDPS, indicating that DISCUSSION nephrotoxicity cannot be attributed to these pretreatments Microsomal enzyme inducers and inhibitors alter NDPS alone. The potentiation of NDPS toxicity that occurred with toxicity (Rankin etal., 1987); however, little is known about PB and ARO induction correlated with changes in the in Downloaded from toxsci.oxfordjournals.org by guest on July 13, 2011 NDPS METABOLISM AND NEPHROTOXICITY 123 vitro metabolic profile of NDPS. In fact, these two pretreat- ABT, a selective, suicide inhibitor of P450 (Mugford et al, ments enhanced formation of the nephrotoxic metabolites 1992), to prevent NDPS metabolism and nephrotoxicity in (2-NDHSA, NDHS, and 3-NDHSA) by the hepatocytes. A rats. BUN levels and kidney weights were elevated in non- similar increase in oxidative metabolism of NDPS by PB- pretreated rats that received a nephrotoxic dose (0.4 mmol/ induced rat liver homogenates was previously described by kg) of NDPS. Diuresis, loss of body weight, proteinuria, and Griffin et al. (1996). A novel, polar metabolite was produced glucosuria were also seen in animals that received this dose. by the ARO-induced cells; however, the role (if any) of this These changes are comparable to prior reports (Rankin, metabolite in NDPS-induced nephrotoxicity is not known. 1982; Rankin et al, 1984, 1985; Kellner-Weibel et al., The ability of SKF 525A to suppress formation of the un- 1995). We also found that ABT pretreatment effectively known and nephrotoxic metabolites by the PB- and ARO- inhibited conversion of NDPS to nephrotoxic metabolites by induced cells indicates that cytochrome(s) P450 is involved the liver cells and prevented NDPS toxicity in vivo. Thus, in their formation (Testa and Jenner, 1981). Overall, these there was an association between inhibition of P450-medi- results indicate that the PB- and ARO-inducible isozymes ated NDPS metabolism in vitro and protection against neph- of P450 are capable of converting NDPS to nephrotoxic rotoxicity. metabolites. In conclusion, we have found that modulation of NDPS Pretreatment with 3-MC or acetone was previously shown metabolism by isolated liver cells correlates closely with to slightly attenuate NDPS-induced nephrotoxicity in rats changes in NDPS-induced renal damage in rats. P450 in- and it was suggested that this could be due to induction of ducers that increase oxidative metabolism of this compound alternative metabolic pathways (Rankin et al, 1987; Lo et in vitro also potentiate its nephrotoxicity in vivo. Inhibition al., 1987). 3-MC, a polycyclic aromatic hydrocarbon, in- of P450-mediated NDPS metabolism protected against neph- duces P450 1A1/1A2 in rat liver (Guengerich et al, 1982), rotoxicity. The results obtained with the different enzyme whereas acetone is an inducer of P450 2E1 (Ryan et al., inducers suggest that only certain P450 isozymes (constitu- 1985; Thomas et al., 1987). Aside from some diuresis and tive and 2B family) can convert NPDS to its nephrotoxic proteinuria, we found little evidence of kidney damage in metabolites. rats that received a nontoxic dose (0.2 mmol/kg) of NDPS following 3-MC pretreatment. These results are somewhat ACKNOWLEDGMENTS different than those reported by Rankin et al. (1987) and may be due to the fact that we used a different induction This publication was made possible by Grant ES05189 from the National Institute for Environmental Health Sciences, NIH, and a Thomas Jefferson protocol (three doses of 3-MC instead of one). Pretreatment Fellowship. The authors thank Dr. Bruce A. Mico (Hoffman-LaRoche, with INH, which, like acetone, is an inducer of P450 2E1 Inc.) for providing the ABT. (Ryan et al., 1985; Thomas et al., 1987), also failed to po- tentiate NDPS nephrotoxicity. Furthermore, we found that REFERENCES 3-MC or INH pretreatment did not enhance the conversion of NDPS to nephrotoxic metabolites in vitro. One possible Barrett, M. C , Cashman, S. J., and Moss, J. (1983). Experimental interstitial explanation for these observations is that induction of the renal fibrosis in rats: Nephritis induced by A'-(3,5-dichlorophenyl)succini- appropriate P450 isozymes was not achieved in the 3-MC- mide. Br. J. Exp. Pathol. 64, 425-436. or INH-pretreated rats; however, hepatocyte and microsomal Fujinami, A., Ozaki, T., Nodera, K., and Tanaka, K. (1972). Studies on P450 levels were elevated following pretreatment with 3- biological activity of cyclic imide compounds: Part II. 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