1H NMR spectroscopic and histopathological studies on propyleneimine-induced renal papillary necrosis in the rat and the multimammate desert mouse (Mastomys natalensis).

The renal papillary toxin, propyleneimine (PI), was administered at 20 or 30 microliters/kg i.p. to male Sprague Dawley (SD) rats (n = 5), Fischer 344 (F344) rats (n = 4), and to multimammate desert mice (Mastomys natalensis, n = 4). Urine was collected at time points up to 4 days p.d. and the toxicological response of the different animal models to PI compared using 1H NMR spectroscopy of urine, renal histopathology, and urinary assays for alkaline phosphatase (ALP), lactate dehydrogenase (LDH), and gamma-glutamyl transpeptidase (gamma GT). The renal papillae of both F344 and SD rats showed extensive necrotic lesions 4 days post-dosing and in some cases sloughing of the papilla. However, only slight renal papillary necrosis (RPN) was observed in Mastomys treated with 20 microliters/kg PI and, although slight to moderate damage was observed at 30 microliters/kg, PI-treated Mastomys showed substantially less RPN than either group of PI-treated rats. 1H NMR urinalysis showed that PI treatment caused a decrease in the urinary concentrations of succinate (0-24 hr p.d.) and citrate (24-48 hr p.d.) and an increase in creatine (0-48 hr p.d.) in all animal models. Trimethylamine-N-oxide (24-48 hr) and 2-oxoglutarate concentrations decreased initially following the administration of PI and then rose above control levels. The 1H NMR-detected urinary biochemical effects of PI in all three models were similar. However, taurine concentrations were elevated in the urine of Mastomys following PI treatment, perhaps indicating a degree of liver damage, whereas taurinuria was not seen in either SD or F344 rats. These observations are discussed in relation to the potential mechanism of PI-toxicity.

Comparative biochemical effects of low doses of mercury II chloride in the F344 rat and the multimammate mouse (Mastomys natalensis).

The biochemical effects and comparative nephroxicity of mercury II chloride (HgCl2) dosed at 0.75 mg/kg i.p. was investigated in the Fisher 344 rat (F344) and Mastomys natalensis using high resolution 1H nuclear magnetic resonance (NMR) spectroscopy of urine, histopathology and clinical chemical techniques. The effects of HgCl2 treatment were followed for up to 4 days post-dosing (p.d.). In F344 rats there was extensive proximal tubular damage and renal cortical necrosis together with elevated levels of urinary gamma-glutamyl transpeptidase (gamma GT), alkaline phosphatase (ALP) and lactate dehydrogenase (LDH). The 1H NMR spectra of urine obtained from Hg-treated F344 rats also showed increased levels of glucose, alanine, lactate, valine and hippurate (0-48h p.d.) with decreased levels of citrate, succinate and 2-oxoglutarate (24-48h p.d.). Mastomys were found to be highly resistant to HgCl2 toxicity at 0.75 mg/kg and the histological appearance of the renal cortex of treated animals was virtually identical to controls. There were no elevations in urinary ALP, gamma GT and LDH activities in HgCl2-treated Mastomys and there were no biochemical abnormalities in low MW components of Mastomys urine following HgCl2-treatment, as shown by 1H NMR spectroscopy. Urinary gamma GT activity was found to be much higher in F344 rats than Mastomys. Since gamma GT activity is involved in the tubular reabsorption of Hg2+, the lower levels of gamma GT in Mastomys might partially account for the lower toxicity of Hg2+ in this species.

A study of the nephrotoxicity of three cephalosporins in rabbits using 1H NMR spectroscopy.

Male New Zealand White rabbits received a single intravenous injection of 125 mg/kg cephaloridine, 500 mg/kg cefoperazone or 1000 mg/kg cephalothin. Histological examination of kidneys at 48 h post-dose confirmed the presence of bilateral necrosis of the proximal convoluted tubules in the cephaloridine-treated animals. 1H-NMR urinalysis of cephaloridine-treated rabbits detected drug-related resonances, decreased hippurate and increased glucose at 0-24 h post-dose accompanied by elevated levels of lactate, glycine, citrate, glutamine/glutamate and alanine at 24-48 h post-dose. No histopathological changes were observed following administration of cefoperazone or cephalothin. 1H-NMR spectra of urine collected from these animals showed drug-related resonances and decreased hippurate levels at 0-24 h post-dose, and increased glucose levels at 24-48 h post-dose. Analysis of urine by conventional clinical-chemistry failed to reveal any statistically significant differences between the treatment groups. Under the conditions of this study, the nephrotoxic effects of cephaloridine and the minimal effects of cefoperazone and cephalothin could be clearly distinguished by 1H-NMR urinalysis but not by conventional urinalysis.

Comparative studies on the nephrotoxicity of 2-bromoethanamine hydrobromide in the Fischer 344 rat and the multimammate desert mouse (Mastomys natalensis).

Renal papillary necrosis (RPN) was induced in Fischer 344 (F344) rats (n = 4) using 2-bromoethanamine hydrobromide (BEA) dosed at 150 mg/kg, and in multimammate desert mice (Mastomys natalensis) at 150 and 250 mg/kg (n = 4 per group). Control rats and Mastomys were dosed with 0.9% saline (n = 4 per group). Urine was collected at regular intervals for up to 4 days post-dosing and analysed for low MW metabolites using high resolution 1H NMR spectroscopy. The urinary activity of lactate dehydrogenase, gamma-glutamyl transpeptidase and alkaline phosphatase was determined using conventional biochemical assays. On termination, histopathological examination of papillae was performed showing the development of extensive lesions in F344 rats at 150 mg/kg BEA. Mastomys appeared much more resistant to BEA and showed normal renal histology at 150 mg/kg and patchy lesions at 250 mg/kg BEA. Enzyme analysis of control urine showed F344 rats to have > 1000% higher gamma-glutamyl transpeptidase activity than Mastomys. 1H NMR spectroscopic analysis showed that BEA caused a substantial decrease in urinary concentrations of succinate and citrate (0-24 h p.d.) and an increase in creatine (0-96 h p.d.) in both animal models. A decrease in the urinary concentration of 2-oxoglutarate with a subsequent increase by 72-96 h p.d. was also noted in both animal models. Glutaric and adipic aciduria were also induced in both F344 rats and Mastomys 0-24 h post-BEA treatment, indicative of an enzyme deficiency in the acyl CoA dehydrogenases. Urinary taurine levels were elevated in Mastomys following the administration of BEA, indicating some degree of liver toxicity. Urinary taurine was not elevated in F344 rats following BEA administration, demonstrating further species difference in BEA toxicity.

The relative hypolipidaemic activity and hepatic effects of ciprofibrate enantiomers in the rat.

The aim of this study was to establish whether the individual enantiomers of racemic ciprofibrate, a potent hypolipidaemic agent and peroxisome proliferator, differ significantly in either pharmacological potency or toxic potential. After a single oral dose to male Fischer F344 rats at dosages below 10 mg/kg, S(-) ciprofibrate produced slightly, but statistically significantly, greater reductions in plasma concentrations of cholesterol than R(+) ciprofibrate. Similarly, at low concentrations in F344 rat hepatocyte cultures, S(-) ciprofibrate produced slightly, but statistically significantly, greater inductions of peroxisomal beta-oxidation activity than R(+) ciprofibrate. However, after seven daily doses, the differences in pharmacological effects of the two enantiomers were no longer apparent. Furthermore, in contrast to its effects in vitro, R(+) ciprofibrate produced slightly, but statistically significantly, greater inductions of peroxisomal beta-oxidation activity in vivo than S(-) ciprofibrate. These observations may be possibly explained on the basis that following multiple dosing, plasma concentrations of R(+) ciprofibrate 24 hr post-dose were greater than those of its optical antipode. Thus the slightly greater potency of the S(-) enantiomer after a single dose may have been overcome by the greater plasma concentrations of the less potent enantiomer. Both enantiomers produced similar reductions in plasma concentrations of thyroxine. The data indicate that at low dosages S(-) ciprofibrate is a slightly more potent hypolipidaemic agent after a single dose in rats and a slightly more potent peroxisome proliferator at low concentrations in vitro. However, following multiple dosing, both enantiomers produced changes in plasma concentrations of lipids, hepatic enzyme activities and plasma concentrations of thyroxine which were of comparable magnitude to those produced by the racemate. Since these early changes have been linked mechanistically to the chronic toxicity of the racemate in the rat, it could be predicted that the individual enantiomers of ciprofibrate under conditions employed in chronic safety studies, would produce the same spectrum of rodent toxicity as the racemate.

The role of investigative toxicology in pharmaceutical industry.

In summary, the benefits of a mechanistic toxicology approach include increased understanding of the processes underlying primary pathology, the development of predictive capability, enhanced decision making and improved responsiveness to regulatory agencies. The overall objective of toxicology in the pharmaceutical industry is risk benefit assessment in order to support product registration. This requires both that studies be conducted in accordance with regulatory guidelines and that the biological processes underlying pathology be understood. These should be regarded as inseparable components of the risk assessment process.

Lack of peroxisome proliferation in marmoset liver following treatment with ciprofibrate for 3 years.

The effect of treatment of marmosets with ciprofibrate for 3 years on activities of hepatic enzymes, hepatic histomorphology, and ultrastructure were investigated. Male and female marmosets were dosed with ciprofibrate (2, 10, and 20 mg/kg) by oral gavage once daily for 3 years. No effect on liver weight (adjusted for body weight) or liver morphology was observed. The activities of catalase, glutathione peroxidase, alpha-glycerophosphate dehydrogenase, benzphetamine N-demethylase, and ethoxyresorufin O-deethylase were unaffected by treatment with ciprofibrate. Activity of glutathione transferase was increased in the low dosage group but unaffected in the mid and high dosage groups. Modest increases in activities of peroxisomal beta-oxidation (2.5-fold, maximal), carnitine acetyl transferase (1.7-fold, maximal), and carnitine palmitoyl transferase (2-fold, maximal) were observed. Cytochemical staining and quantitative image analysis failed to indicate any effect on peroxisomal number, size, or volume density. Similarly, there was no increase in lipofuscin deposition. This study provides data on the effects of a potent peroxisome proliferator on primate liver following a dosing period much greater than that used in previously published studies and is further evidence that the marmoset is relatively insensitive to the well-documented effects that ciprofibrate and other peroxisome proliferators have on rat liver.

Nuclear magnetic resonance spectroscopy and pattern recognition analysis of the biochemical processes associated with the progression of and recovery from nephrotoxic lesions in the rat induced by mercury(II) chloride and 2-bromoethanamine.

Nephrotoxic lesions were induced in Fischer 344 rats using HgCl2, a proximal tubular toxin, and 2-bromoethanamine (BEA), a medullary toxin. Biochemical effects of these toxins on urinary composition were observed by high resolution 1H NMR spectroscopy over 9 days after dosing. The onset of, progression of, and recovery from the induced toxic lesions were also followed histopathologically and related to the perturbed urinary biochemistry. Urinary concentrations of 20 endogenous substances were measured simultaneously by NMR at eight time points, to provide a time-related 20-dimensional description of the urinary biochemistry for each rat. Principal components analysis and nonlinear mapping were used to reduce the biochemical parameter spaces for each rat to two or three dimensions for display and classification purposes. An investigation of alternative data-presentation methods was made, and taking interanimal means of the map coordinates at each time point yielded a novel type of metabolic trajectory diagram with which the biochemical abnormalities associated with the HgCl2 and BEA lesions could be related to the progression and recovery phases of the toxic lesions. The time-course trajectories showed characteristically different paths for each toxin. These trajectories allowed the time points at which there were maximum metabolic differences to be determined and provided the visualization of net movements of the treatment group populations in time in relation to interanimal variation. Control animal urine samples subjected to this analysis showed simple clustering, with no evidence of metabolic trajectory. The trajectory for BEA showed different routes for onset of and recovery from toxicity, whereas for HgCl2 the outward trajectory (onset) mapped a space similar to the inward trajectory (recovery phase). This suggests that the NMR-detectable biochemical abnormalities after mercury toxicity mainly reflect the proportions of functional cells lining the nephron, whereas the biochemical abnormalities associated with renal medullary insult probably relate to functional integrity. An examination has been made for those metabolites that are most responsible for defining the trajectories, i.e., the discrimination of renal cortical and medullary toxicity from each other and from controls. These discriminatory metabolites (using paired t test, p < 0.001) included valine, taurine, trimethylamine N-oxide, and glucose for HgCl2 and acetate, methylamine, dimethylamine, lactate, and creatine for BEA, whereas citrate, succinate, N-acetyl resonances from as yet unidentified metabolites, hippurate, alanine, and 2-oxoglutarate played an important role in defining the biochemically perturbed trajectory of both toxins.(ABSTRACT TRUNCATED AT 400 WORDS)

Species differences in ciprofibrate induction of hepatic cytochrome P450 4A1 and peroxisome proliferation.

Six species (CD-1 mouse, Fischer 344 rat, Syrian golden hamster, Duncan-Hartley guinea pig, half-lop rabbit and marmoset monkey) were treated orally with ciprofibrate, a potent oxyisobutyrate hypolipidaemic drug for 14 days. A dose-dependent liver enlargment was observed in the mouse and rat and at the high dose level in the hamster. A marked dose-dependent increase in the 12-hydroxylation of lauric acid was observed in the treated mouse, hamster, rat, and rabbit, associated with a concomitant elevation in the specific content of cytochrome P-450 4A1 apoprotein, determined by an ELISA technique. Similarly, in these responsive species, an increase in mRNA levels coding for cytochrome P450 4A1 was observed. Lauric acid 12-hydroxylation was unchanged in the guinea pig and marmoset after ciprofibrate pretreatment, and cytochrome P-450 4A1 was not detected immunochemically in liver microsomes from these latter species. In the untreated mouse, hamster, rat, and rabbit, the 12-hydroxylation of lauric acid was more extensive than the 11-hydroxylation, whereas in the guinea pig and marmoset the activity ratios were reversed, with 11-hydroxylation predominating. Peroxisomal fatty acid beta-oxidation was markedly induced in the mouse, hamster, rat, and rabbit on treatment at the higher dose level (39-, 3-, 13- and 5-fold, respectively) and was slightly increased in the marmoset (2-fold), yet was unchanged in the guinea pig following treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative induction of cytochrome P450IVA1 and peroxisome proliferation by ciprofibrate in the rat and marmoset.

Chronic ciprofibrate administration resulted in distinct differences in hepatic responses between the two species examined. In the rat, hepatomegaly was observed with the coordinate induction of carnitine acetyltransferase, peroxisomal beta-oxidation and cytochrome P450IVA1 activities. The latter induction of cytochrome P450IVA1-dependent fatty acid hydroxylase activity was specific to this cytochrome P450 sub family, as ciprofibrate pretreatment resulted in an inhibition of the enzyme activities associated with the cytochrome P450 IIB and IA sub-families. Induction of mitochondrial enzymes were also noted in the rat, but at a substantially lower level than the microsomal and peroxisomal enzyme changes noted above. The majority of these enzyme changes were reversible in the rat after a 4-week, inducer-free period. In contrast, the marmoset displayed a different pattern of enzyme changes in response to ciprofibrate and at the high dose level, inhibition of microsomal fatty acid hydroxylase activity was observed in addition to no change in carnitine acetyltransferase activity. Although peroxisomal beta-oxidation activity was induced in the marmoset, the specific activity was 10-fold lower than in the rat, concomitant with only minimum changes in the liver: body weight ratio. Taken collectively, our data have demonstrated that the marmoset is relatively refractory to ciprofibrate-induced liver enzyme changes with the implication that the extrapolation of the associated hepatotoxicity clearly documented in rodents must be viewed with extreme caution in non-human primates.

Hepatic induction potency of hypolipidaemic drugs in the rat following long-term administration: influence of different dosing regimens.

1. The effects of different dosing regimens of three hypolipidaemic, peroxisome-proliferator drugs on hepatic enzymes in the Fischer rat following 26 weeks treatment have been studied. 2. In study 1, with once-daily dosing (dose levels based on comparative antisecretory activity), the liver/body weight ratio and peroxisomal beta-oxidation were significantly increased in the order: ciprofibrate greater than bezafibrate greater than clofibric acid. Glutathione peroxidase activity was decreased to 65% and 77% control after treatment with ciprofibrate and bezafibrate, respectively, but not after treatment with clofibric acid. 3. In study 2, dosing regimens were adjusted to compensate for the different drug pharmacokinetic profiles in rat, with clofibric acid and bezafibrate administered twice daily and ciprofibrate once every 48 h. Liver enlargement and increases in peroxisomal beta-oxidation were similar with all three drugs when compensation for differences in drug clearance was made. Glutathione peroxidase activity was decreased to similar extents by all three compounds. 4. The induction profiles of these hypolipidaemic drugs, largely different with once-daily dosing, were shown to be similar after adjusting the frequency of dosing with respect to drug half-life.

The importance of pharmacokinetic and receptor studies in drug safety evaluation.

The importance of pharmacokinetic and receptor studies in the preclinical and clinical safety evaluation of candidate drugs is reviewed with reference to a number of recently developed drugs. Different aspects of the relationships between pathways of metabolism, pharmacokinetics, receptor interactions, and drug toxicity are illustrated. The failure of animal toxicity studies to predict drug toxicity in humans, due to species differences in metabolism and pharmacokinetics, is illustrated by reference to the anti-inflammatory antiviral terpenoid carbenoxolone, the antiasthmatic candidate drug FPL 52757, and the cardiotonic drug amrinone. The false prediction of adverse effects in man from toxicity manifested in experimental animals, due to species differences in pharmacokinetics or receptor activities, is exemplified with reference to the antiepileptic valproic acid, the hypolipidemic drug ciprofibrate, the antipeptic ulcer drug, omeprazole, and the progestogen lynestrenol. Finally, the importance of adequate, repeat-dose, clinical pharmacokinetic studies in patients as distinct from healthy volunteers to evaluate any effect of the disease state, in the elderly and the young to examine the effects of age, and in sufficiently large populations to detect genetic anomalies and idiosyncrasies is illustrated by reference to the anti-rheumatoid drug benoxaprofen, the antiangina drug perhexiline, and the diuretic tienilic acid.

A comparison of the effects of three positive inotropic agents (amrinone, milrinone and medorinone) on platelet aggregation in human whole blood.

Amrinone, milrinone and medorinone inhibit platelet aggregation in human whole blood. They are particularly potent inhibitors of arachidonic acid induced aggregation, inhibiting by 50% (IC50) at concentrations of 1.5 microM (milrinone), 7.5 microM (medorinone) and 48 microM (amrinone). Each drug was less potent at inhibiting ADP and collagen-induced aggregation. The rank order for inhibition of arachidonic acid - induced aggregation correlated well with the rank order of cyclic AMP phosphodiesterase inhibition for these drugs when compared to the response of a reference cAMP phosphodiesterase inhibitor (CI-930) and a reference cGMP phosphodiesterase inhibitor (M & B 22948). Since inhibition of platelet aggregation in vitro occurred at clinically relevant concentrations, it is evident that these agents have potentially beneficial antithrombotic properties.

Effects of biliary cannulation and buthionine sulphoximine pretreatment on the nephrotoxicity of para-aminophenol in the Fischer 344 rat.

The effects of a glutathione depletor, buthionine sulphoximine (BSO) and biliary cannulation on the nephrotoxicity of p-aminophenol (PAP) have been investigated in the F344 rat. Pretreatment with BSO completely protected against the nephrotoxicity of a 50 mg/kg dose of PAP, assessed by clinical chemistry, renal histopathology, and 1H-NMR urinalysis. Biliary cannulation partially protects against nephrotoxicity induced by 100 mg/kg PAP. These data suggest that the nephrotoxicity of PAP may be due in part to the formation of a proximate toxic metabolite in the liver which is excreted in the bile, subsequently reabsorbed and transported via the systemic circulation to the kidney where the toxic effects occur.

The comparative pharmacokinetics and gastric toxicity of bezafibrate and ciprofibrate in the rat.

1. The comparative gastric toxicology and pharmacokinetics of two phenoxyisobutyrate derivatives have been evaluated in the Fischer rat. 2. After oral administration of single daily doses for 7 days, the plasma elimination half-life for bezafibrate was rapid (t1/2 of 4-5 h) in comparison to ciprofibrate (t1/2 of 76 h). 3. The area under the plasma drug concentration versus time curve (AUC) 0-24 (micrograms.h/ml +/- SD) for bezafibrate (dose 125 mg/kg per day) was 1553 +/- 334, which was less than half the value of 3748 +/- 358 achieved by ciprofibrate (10 mg/kg per day) after 7 days. 4. Oral administration of ciprofibrate at 10 mg/kg every 48 h produced similar sustained plasma concentrations to those achieved by bezafibrate 125 mg/kg dosed every 12 h. The AUC 0-48 values (micrograms.h/ml +/- SD) achieved were 5124 +/- 450 for bezafibrate compared to 4207 +/- 240 for ciprofibrate. 5. In chronic oral multidose studies with ciprofibrate and bezafibrate, similar gastric toxicity (neuroendocrine cell hyperplasia) occurred in the rat when dose regimens were adjusted to compensate for the pharmacokinetic differences between these two drugs.

The trophic effects of gastrin on fundic neuroendocrine cells of the rat stomach.

The histomorphological effect of multidose administration of 6 mg/kg pentagastrin b.d. for 5 weeks, and 1000 mg/kg sodium bicarbonate b.d. for 13 weeks, on the rat fundic mucosa has been examined. Sodium bicarbonate induced a significant hypergastrinaemia (plasma gastrin concentrations were 370.5 pg/ml in the control versus 642.6 pg/ml in sodium bicarbonate-treated rats after 13 weeks, P less than 0.01). Both treatment regimens induced fundic neuroendocrine cell hyperplasia. The cellular proliferation that occurred following hypergastrinaemia of endogenous or exogenous origin suggests that systemic gastrin concentrations play a major role in the control of fundic neuroendocrine cell populations.