Nephrotoxicity induced by the R- and S-enantiomers of N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS) and their sulfate conjugates in male Fischer 344 rats
Introduction
N-(3,5-Dichlorophenyl)succinimide (NDPS) was developed as an agricultural fungicide in Japan during the early 1970s (Fujinami et al., 1971, Fujinami et al., 1972). Although NDPS was a highly efficacious agent, potential health concerns associated with NDPS exposure have limited its usefulness in agriculture (Heaton, 1980). Nonetheless, NDPS is still available in several countries, including the United States. The primary toxicity induced by exposure to NDPS is nephrotoxicity. Administration of NDPS to male Sprague–Dawley or Fischer 344 rats at intraperitoneal (i.p.) doses of 0.4 mmol/kg or greater results in acute polyuric renal failure and proximal tubular necrosis (Rankin, 1982, Rankin et al., 1984, Rankin et al., 1985). Chronic exposure to NDPS in food (5000 ppm) results in the development of chronic interstitial nephritis in rodents (Sugihara et al., 1975, Barrett et al., 1983).
NDPS is extensively biotransformed in the liver through hydrolysis (ring opening) and oxidation pathways (Fig. 1), and less than 1% of NDPS is excreted unchanged (Ohkawa et al., 1974, Nyarko and Harvison, 1995, Griffin and Harvison, 1998). The NDPS metabolite that results from hydrolysis, N-(3,5-dichlorophenyl)succinamic acid (NDPSA), is a weakly nephrotoxic metabolite at i.p. doses up to 1.0 mmol/kg, but accounts for ∼40% of urinary metabolites (Yang et al., 1985, Griffin and Harvison, 1998). Oxidation of the succinimide ring results in the formation of N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS), which can be hydrolyzed in vivo to form N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (2-NDHSA) and N-(3,5-dichlorophenyl)-3-hydroxysuccinamic acid (3-NDHSA). Although NDHS is not detected in urine, 2- and 3-NDHSA constitute ∼30 and 20% of urinary NDPS metabolites, respectively (Griffin and Harvison, 1998). Both NDHS and 2-NDHSA are at least four times more potent than NDPS as a nephrotoxicant in rats with 3-NDHSA being approximately equipotent to NDPS in inducing nephrotoxicity (Hong et al., 1998, Rankin et al., 1989, Rankin et al., 1991a, Rankin et al., 1991b, Rankin et al., 1994). When 2-NDHSA undergoes decarboxylation, N-(3,5-dichlorophenyl)malonamic acid (DMA), a non-nephrotoxic metabolite that constitutes ∼10% of urinary NDPS metabolites, is formed (Rankin et al., 1988, Griffin and Harvison, 1998). Oxidation of the phenyl ring of NDPS also results in minor non-nephrotoxic metabolites (Harvison et al., 1992). Thus, the primary route of bioactivation of NDPS to a nephrotoxic metabolite(s) initially involves oxidation of the succinimide ring to form NDHS and 2-NDHSA.
Previous studies have determined that Phase II conjugates (glucuronide and sulfate conjugates) of NDPS metabolites contribute to the primary mechanism of NDPS nephrotoxicity (Hong et al., 1999a, Hong et al., 1999b, Hong et al., 1999c, Hong et al., 1999d, Rankin et al., 1997). Recently, Cui et al. (2005) reported the detection of small amounts of the O-glucuronide and O-sulfate conjugates of 2-/3-NDHSA in the urine of rats treated with NDPS. It was determined that these conjugates were formed by conjugation of NDHS followed by hydrolysis to the NDHSA conjugates. While earlier studies had demonstrated that N-(3,5-dichlorophenyl)-2-hydroxysuccinimide-O-sulfate (NSC; Fig. 2) was a nephrotoxicant in vitro (Rankin et al., 2001a), Cui et al. (2005) also found that the NDHS conjugates are highly reactive and readily form glutathione-derived conjugates. Thus, the mechanism of NDPS nephrotoxicity appears to be linked to the formation of these reactive metabolites.
An asymmetric carbon atom is present at the 2-position of the succinimide ring in NDHS and in the ethylene bridge of 2-NDHSA. Therefore, two stereoisomers, the R- and S-enantiomers, for NDHS and 2-NDHSA are possible. In a previous study, it was noted that the two enantiomers of 2-NDHSA exhibited very different nephrotoxic potentials (Rankin et al., 2001b). The S-(−)-2-NDHSA enantiomer was a potent nephrotoxicant, while the R-(+)-2-NDHSA enantiomer was a weak nephrotoxicant at the doses tested. Thus, for at least one nephrotoxicant metabolite of NDPS, there is enantiomer selective nephrotoxicity. However, it is not know if this stereoselectivity for nephrotoxic potential applies to NDHS or if the mechanism for this enantiomeric selectivity has a renal or extra-renal origin.
The purpose of this study was to examine the nephrotoxic potential of the two enantiomers of NDHS (Fig. 2) in vivo using male Fischer 344 rats as the animal model. The two enantiomers of NSC (Fig. 2) were examined in vitro using freshly isolated renal cortical cells from male Fischer 344 rats to determine if, at least for NSC, the stereoselectivity in the nephrotoxic response derived from a renal or extra-renal mechanism.
Section snippets
Experimental animals
Male (200–250 g) Fischer 344 rats were obtained from Hilltop Lab Animals Inc. (Scottdale, PA, USA) and housed in standard plastic animal cages (four rats/cage) prior to use. Animal holding and experimental rooms had a controlled light period (on 06:00 h, off 18:00 h), humidity (40–55%) and temperature (21–23 °C). At least 1 week was allowed for acclimation to the animal facilities prior to initiation of experiments. All animals were maintained and handled in agreement with the Institutional Guide
R- and S-NDHS nephrotoxicity in vivo
Rats treated with R-NDHS (0.1 mmol/kg) exhibited little evidence of nephrotoxicity. Food and water intake (Table 1), urine volume (Fig. 3), urinary protein (Fig. 4) and urinary glucose (Table 2) excretion and kidney weight (Fig. 5) were not altered in the R-NDHS (0.1 mmol/kg) treatment group. BUN concentration decreased slightly on day 2 relative to day 0 value for the treated group and the day 2 value for the control group (Table 2), but this change is not considered to be toxicologically
Discussion
Biological activity or pharmacodynamic properties of drugs and toxicants can be stereoselective in nature (Mathison et al., 1995). One example of this effect is that while the anti-inflammatory activity of the non-steroidal anti-inflammatory drugs resides mainly in the S-(+)-enantiomers (de la Lastra et al., 2000), R-ibuprofen stereoselectively inhibits beta-oxidation of lipids (Browne et al., 1999). An example where toxicants exhibit stereoselective properties can be found with
Conflict of interest
None of the authors have any conflict of interest related to this work.
Acknowledgements
This work was supported by NIH grant DK31210. The authors would like to thank Amanda Casto and Tim Crislip for their technical assistance. Histological examination of tissue was performed by Dr. Triest at the Veterans Affairs Medical Center, Huntington, WV, USA.
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