Peter J. Harvison

The effects of fructose on adenosine triphosphate depletion following mitochondrial dysfunction and lethal cell injury in isolated rat hepatocytes

Mitochondrial injury in aerobic mammalian cells is associated with a rapid depletion of adenosine triphosphate (ATP) which occurs prior to the onset of lethal cell injury. In this report, the relationships between ATP depletion and lethal cell injury were examined in rat hepatocytes using oligomycin as a model mitochondrial toxicant and fructose as an alternative carbohydrate source for glycolysis. Oligomycin was more potent in causing lethal cell injury in hepatocytes isolated from fasted animals than cells from fed animals. The onset of cell injury (leakage of lactate dehydrogenase) in cells from fed animals correlated with the depletion of stored glycogen and ATP. The degree and time course profile of oligomycin-induced ATP depletion could be duplicated with 50 mM fructose alone in hepatocytes from fasted animals; however, fructose did not cause lethal cell injury. Oligomycin caused marked accumulation of adenosine monophosphate (AMP) and inorganic phosphate (Pi) and a conservation of adenine nucleotides. In contrast, fructose (50 mM) caused a decrease in Pi, no persistent change in AMP, and a depletion of the adenine nucleotide pool. Fructose, at concentrations greater than 1.0 mM, protected hepatocytes from oligomycin-induced toxicity. Blockade of mitochondrial ATP synthesis with oligomycin resulted in massive ATP depletion. In the presence of oligomycin, 5.0 mM fructose maintained cellular ATP content similar to that of control cells, whereas 50 mM fructose did not, demonstrating the biphasic effect of increasing fructose concentrations on cellular ATP content. Fructose-induced protection of hepatocytes from oligomycin toxicity was due to glycolytic fructose metabolism as hepatocytes incubated with iodoacetate (30 microM), fructose, and oligomycin had reduced viability and ATP content. In conclusion, interruption of mitochondrial ATP synthesis leads to marked ATP depletion and lethal cell injury. Cell injury is clearly not due to ATP depletion alone since increased glycolytic ATP production from either glycogen or fructose can maintain cell integrity in the absence of mitochondrial ATP synthesis and at low cellular ATP levels.

Potential metabolism and cytotoxicity of N-(3,5-dichlorophenyl)succinimide and its hepatic metabolites in isolated rat renal cortical tubule cells

N-(3,5-Dichlorophenyl)succinimide (NDPS) is an agricultural fungicide and antimicrobial agent that produces nephrotoxicity in rats. The contribution of the kidney, if any, to the mechanism of toxicity of NDPS is not known. Therefore, the ability of isolated renal cortical tubule cells to metabolize NDPS and some of its known hepatic metabolites was studied. The cytotoxic potential of these compounds was also assessed. Renal cortical tubule cells were isolated by collagenase digestion and were incubated with the test compounds (2 mM) for 3 h. Metabolite formation was monitored by reversed phase HPLC and cell viability was assessed using trypan blue exclusion. The isolated kidney cells do not appear to metabolize NDPS or any of its known hepatic metabolites. In addition, none of these compounds were directly cytotoxic to the renal cells. However, the cells were susceptible to mercuric chloride (1 mM) and chloroform (125 or 200 mM). Intracellular glutathione levels were unaltered by the presence of NDPS in the incubations. These results suggest that NDPS and its metabolites are not directly toxic to the kidney and are not converted into the ultimate nephrotoxic species by the kidney. Extrarenal metabolism may, therefore, be critical to the expression of NDPS-induced nephrotoxicity.

Nephrotoxic potential of N-(3,5-dichloro-4-hydroxyphenyl)succinimide and N-(3,5-dichloro-4-hydroxyphenyl)succinamic acid in Fischer-344 rats

The ultimate nephrotoxicant species following administration of the agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) has yet to be determined. The purpose of this study was to examine the nephrotoxic potential of two potential metabolites of NDPS, N-(3,5-dichloro-4-hydroxyphenyl)-succinimide (NDHPS) and N-(3,5-dichloro-4-hydroxyphenyl)succinamic acid (NDHPSA). Male Fischer-344 rats (4 rats/group) were administered a single intraperitoneal injection of NDHPS or NDHPSA (0.2 or 0.4 mmol/kg) or vehicle and renal function was monitored at 24 and 48 h. Neither compound induced marked changes in renal function or morphology. These results suggest that NDHPS and NDHPSA do not contribute significantly to NDPS-induced nephrotoxicity.

Determination of the site of glucuronidation in an N-(3,5-dichlorophenyl)succinimide metabolite by electrospray ionization tandem mass spectrometry following derivatization to picolinyl esters.

Derivatization using 3-pyridylcarbinol coupled with liquid chromatography electrospray ionization tandem mass spectrometry (LC/MS/MS) was used to characterize a novel Phase II metabolite of the nephrotoxic agricultural fungicide, N-(3,5-dichlorophenyl)succinimide (NDPS). A glucuronide conjugate of N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (2-NDHSA) was identified in the urine from a rat dosed with [14C]NDPS. However, 2-NDHSA contains an aliphatic hydroxyl group and a carboxylic acid group, both of which are potential sites for glucuronidation. Mass spectrometry alone was unable to distinguish between these possibilities. Since the position of glucuronidation may be important in the mechanism of NDPS-induced nephrotoxicity, chemical derivatization in conjunction with mass spectrometry was used to characterize the glucuronide. The 2-NDHSA glucuronide conjugate was isolated from rat urine, derivatized with 3-pyridylcarbinol, and the derivatized metabolite was then analyzed by LC/MS/MS. Two known NDPS metabolites, 2-NDHSA and N-(3,5-dichlorophenyl)succinamic acid (NDPSA), were also isolated from rat urine and derivatized similarly. 3-Pyridinylcarbinol reacted rapidly with the carboxylic acid groups and formation of the picolinyl esters increased the ionization potential under positive ion conditions. The urinary glucuronide of 2-NDHSA was identified as an alcohol-linked glucuronide by examination of the molecular ions and the collision-induced dissociation (CID) product ion spectra of the derivatized products. When used in combination with mass spectrometry, derivatization of carboxylic acids with 3-pyridylcarbinol provided useful mass fragmentations and is a rapid way to obtain structural information about the position of glucuronidation of NDPS metabolites.

Nephrotoxic and hepatotoxic potential of imidazolidinedione-, oxazolidinedione- and thiazolidinedione-containing analogues of N-(3,5-dichlorophenyl)succinimide (NDPS) in Fischer 344 rats ☆

Nephrotoxicity of the agricultural fungicide N-(3,5-dichlorophenyl)succinimide (NDPS) in rats is believed to involve metabolism on the succinimide ring. To further investigate this hypothesis, we synthesized and tested the following NDPS analogues, which contain other cyclic imide rings and may therefore be metabolized differently than NDPS: 3-(3,5-dichlorophenyl)-2,4-oxazolidinedione (DCPO), 3-(3,5-dichlorophenyl)-2,4-imidazolidinedione (DCPI), 3-(3,5-dichlorophenyl)-1-methyl-2,4-imidazolidinedione (DCPM) and 3-(3,5-dichlorophenyl)-2,4-thiazolidinedione (DCPT). Male Fischer 344 rats were administered DCPO, DCPI, DCPM, DCPT (0.6 or 1.0 mmol/kg, i.p. in corn oil), NDPS (0.6 mmol/kg, i.p. in corn oil) or corn oil (4 ml/kg). As evidenced by diuresis, proteinuria, elevated blood urea nitrogen levels, increased kidney weights and proximal tubular damage, NDPS produced severe nephrotoxicity in the rats. In contrast, DCPO, DCPI, DCPM and DCPT were mild nephrotoxicants. None of the compounds elevated serum alanine transferase activity or liver weights in the rats, however DCPT produced centrilobular necrosis. These experiments confirm that NDPS-induced nephrotoxicity is critically dependent on the presence of the succinimide ring. Furthermore, replacement of the succinimide ring with a thiazolidinedione ring produced a more pronounced effect on the liver than on the kidney. Liver damage has been reported in type II diabetic patients taking troglitazone, rosiglitazone and pioglitazone. Since these compounds also contain a thiazolidinedione ring, DCPT may be useful for investigating the role of this structural feature in hepatotoxicity.

The effect of aromatic fluorine substitution on the nephrotoxicity and metabolism of N-(3,5-dichlorophenyl)succinimide in fischer 344 rats

N-(3,5-Difluorophenyl)succinimide (DFPS) is a non-toxic analogue of the nephrotoxic fungicide N-(3,5-dichlorophenyl)succinimide (NDPS). Although NDPS must be metabolized to produce renal damage, the metabolic fate of DFPS is unknown. These studies were therefore designed to examine the nephrotoxic potential of putative DFPS metabolites and to determine if DFPS is metabolized differently from NDPS. Male Fischer-344 rats were administered (1.0 mmol/kg. i.p. in corn oil) DFPS, N-(3,5-difluorophenyl)succinamic acid (DFPSA), N-(3,5-difluorophenyl)-2-hydroxysuccinimide (DFHS), N-(3,5-difluorophenyl)-2- or -3-hydroxysuccinamic acids (2- and 3-DFHSA, respectively), N-(3,5-difluoro-4-hydroxyphenyl)succinimide (DFHPS). N-(3,5-difluoro-4-hydroxyphenyl) succinamic acid (DFHPSA) or corn oil only (1.2 ml/kg). Although some of the compounds produced changes in renal function and histology, these alterations were not indicative of irreversible kidney damage. DFPSA, 2-DFHSA, 3-DFHSA and DFHPSA were detected in the urine of rats 3 h after administration of 0.2 mmol/kg [14C]DFPS. The same metabolites were produced by isolated rat hepatocytes, but not by renal proximal tubule cells. Formation of the oxidative metabolites in vitro was prevented by the cytochrome P450 inhibitor 1-aminobenzotriazole. It appears that DFPS undergoes hepatic biotransformation similar to NDPS and that some of its metabolites have reversible effects on renal proximal tubules.

In vitro metabolism of the nephrotoxicant N-(3,5-dichlorophenyl)succinimide in the Fischer 344 rat and New Zealand white rabbit

1. The nephrotoxicant N-(3,5-dichlorophenyl)succinimide (NDPS) underwent nonenzymatic hydrolysis to N-(3,5-dichlorophenyl)succinamic acid (NDPSA) in buffer, rat liver and kidney homogenates, and rabbit liver homogenates. 2. In the presence of NADPH, rat liver homogenates converted NDPS to NDPSA and N-(3,5-dichlorophenyl)-2-hydroxysuccinamic acid (2-NDHSA). 3. Using liver homogenates from the phenobarbital (PB)-pretreated rat, 2-NDHSA production was increased 5-fold, and the metabolites N-(3,5-dichlorophenyl)-2-hydroxysuccinimide (NDHS) and N-(3,5-dichlorophenyl)-3-hydroxysuccinamic acid (3NDHSA) were also detected. Formation of these latter metabolites was suppressed by CO or omission of NADPH. No hydroxylated metabolites were detected when NDPSA was incubated with PB-induced rat liver homogenates. 4. Oxidative metabolites were not produced when NDPS was incubated with kidney homogenates from the control or PB-pretreated rat. 5. NDHS underwent rapid hydrolysis in buffer to yield 2-NDHSA and 3-NDHSA. 6. Rabbit liver homogenates converted NDPS to NDPSA, 3,5-dichloroaniline (DCA), and succinic acid (SA). Production of DCA and SA was inhibited by the amidase inhibitor bis-p-nitrophenyl phosphate. Oxidative metabolism did not occur in rabbit tissue. 7. These experiments demonstrate that a PB-inducible form of rat liver P450 converts NDPS to NDHS, which then undergoes hydrolysis to 2-NDHSA and 3-NDHSA. An alternative route of production for 2-NDHSA and 3-NDHSA, via hydroxylation of NDPSA, does not occur. In rabbit liver NDPS metabolism was primarily amidase-mediated.

Effect of structural modifications on 3-(3,5-dichlorophenyl)-2,4-thiazolidinedione-induced hepatotoxicity in Fischer 344 rats.

Glitazones, used for type II diabetes, have been associated with liver damage in humans. A structural feature known as a 2,4‐thiazolidinedione (TZD) ring may contribute to this toxicity. TZD rings are of interest since continued human exposure via the glitazones and various prototype drugs is possible. Previously, we found that 3‐(3,5‐dichlorophenyl)‐2,4‐thiazolidinedione (DCPT) was hepatotoxic in rats. To evaluate the importance of structure on DCPT toxicity, we therefore studied two series of analogs. The TZD ring was replaced with: a mercaptoacetic acid group {[[[(3,5‐dichlorophenyl)amino]carbonyl]thio]acetic acid, DCTA}; a methylated TZD ring [3‐(3,5‐dichlorophenyl)‐5‐methyl‐2,4‐thiazolidinedione, DPMT]; and isomeric thiazolidinone rings [3‐(3,5‐dichlorophenyl)‐2‐ and 3‐(3,5‐dichlorophenyl)‐4‐thiazolidinone, 2‐DCTD and 4‐DCTD, respectively]. The following phenyl ring‐modified analogs were also tested: 3‐phenyl‐, 3‐(4‐chlorophenyl)‐, 3‐(3,5‐dimethylphenyl)‐ and 3‐[3,5‐bis(trifluoromethyl)phenyl]‐2,4‐thiazolidinedione (PTZD, CPTD, DMPT and DFMPT, respectively). Toxicity was assessed in male Fischer 344 rats 24 h after administration of the compounds. In the TZD series only DPMT produced liver damage, as evidenced by elevated serum alanine aminotransferase (ALT) activities at 0.6 and 1.0 mmol kg−1 (298.6 ± 176.1 and 327.3 ± 102.9 Sigma‐Frankel units ml−1, respectively) vs corn oil controls (36.0 ± 11.3) and morphological changes in liver sections. Among the phenyl analogs, hepatotoxicity was observed in rats administered PTZD, CPTD and DMPT; with ALT values of 1196.2 ± 133.6, 1622.5 ± 218.5 and 2071.9 ± 217.8, respectively (1.0 mmol kg−1 doses). Morphological examination revealed severe hepatic necrosis in these animals. Our results suggest that hepatotoxicity of these compounds is critically dependent on the presence of a TZD ring and also the phenyl substituents. Copyright

Effect of gender, dose, and time on 3-(3,5-dichlorophenyl)-2,4-thiazolidinedione (DCPT)-induced hepatotoxicity in Fischer 344 rats.

1. The thiazolidinedione ring present in drugs available for type II diabetes can contribute to hepatic injury. Another thiazolidinedione ring-containing compound, 3-(3,5-dichlorophenyl)-2,4-thiazoli-dinedione (DCPT), produces liver damage in rats. Accordingly, the effects of gender, dose, and time on DCPT hepatotoxicity were therefore evaluated. 2. Male rats were more sensitive to DCPT (0.4–1.0 mmol kg−1 by intraperitoneal administration) as shown by increased serum alanine aminotransferase levels and altered hepatic morphology 24 h post-dosing. Effects in both genders were dose dependent. In males, DCPT (0.6 mmol kg−1) produced elevations in alanine aminotransferases and changes in liver h after dosing that progressively worsened up to 12 h. DCPT-induced renal effects were mild. 3. It is concluded that male rats are more susceptible to DCPT hepatotoxicity and that damage occurs rapidly. DCPT primarily affects the liver and can be a useful compound to investigate the role of the thiazolidinedione ring in hepatic injury. However, the gender dependency and rapid onset of DCPT hepatotoxicity require further investigation.

Role of biotransformation in 3-(3,5-dichlorophenyl)-2,4-thiazolidinedione-induced hepatotoxicity in Fischer 344 rats

Cytochrome P450 (CYP)-mediated metabolism in the thiazolidinedione (TZD) ring may contribute to the hepatotoxicity of the insulin-sensitizing agents such as troglitazone. We were interested in determining if biotransformation could also be a factor in the liver damage associated with another TZD ring containing compound, 3-(3,5-dichlorophenyl)-2,4-thiazolidinedione (DCPT). Therefore, hepatotoxic doses of DCPT (0.6 or 1.0 mmol/kg, i.p.) were administered to male Fischer 344 rats after pretreatment with vehicle, 1-aminobenzotriazole (ABT, non-selective CYP inhibitor) and troleandomycin (TAO, CYP3A inhibitor). Alternatively, rats were pretreated with vehicle or the CYP3A inducer dexamethasone (DEX) prior to a non-toxic DCPT dose (0.2 mmol/kg, i.p.). Vehicle-, ABT-, TAO- and DEX-only control groups were also run. Toxicity was assessed 24 h after DCPT administration. Both hepatotoxic doses of DCPT induced elevations in serum alanine aminotransferase (ALT) levels that were attenuated by ABT or TAO pretreatment. Liver sections from rats that received vehicle+DCPT revealed areas of gross necrosis and neutrophil invasion, whereas sections from ABT+DCPT and TAO+DCPT rats showed minor changes compared to controls. DEX pretreatment potentiated ALT levels associated with the non-toxic DCPT dose. Furthermore, DEX+DCPT rat liver sections exhibited hepatic injury when compared against rats that received vehicle+DCPT. Blood urea nitrogen levels, urinalysis and kidney morphology were not markedly altered by any combination of pretreatments or treatments. Enzyme activity and Western blotting experiments with rat liver microsomes confirmed the effects of the various pretreatments. Our results suggest that hepatic CYP3A isozymes may be involved in DCPT-induced liver damage in male rats. We believe this is the first report demonstrating that modulation of the biotransformation of a TZD ring-containing compound can alter hepatotoxicity in a common animal model.