Robin M. Walker

Inhibition of mitochondrial respiration in vivo is an early event in acetaminophen-induced hepatotoxicity

Morphological changes in mitochondria are observed early in the course of acetaminophen (AA)-induced hepatotoxicity, and mitochondrial dysfunction has been observed both in vivo and in vitro following exposure to AA. This study examined the early effects of AA exposure in vivo on mitochondrial respiration and evaluated the effectiveness ofN-acetyl-L-cysteine (NAC) in protecting against respiratory dysfunction. Mitochondria were isolated from the livers of fasted, male CD-1 mice 0, 0.5, 1, 1.5 or 2 h after administration of a hepatotoxic dose of AA (750 mg/kg). Glutamate- and succinate-supported mitochondrial respiration were subsequently assessed by polarographic measurement of state 3 (ADP-stimulated) and state 4 (resting) rates of oxygen consumption and determination of the corresponding respiratory control ratios (RCR: state 3/state 4) and ADP:O ratios. Hepatotoxicity was assessed histologically and by measuring plasma alanine aminotransferase (ALT) activity. The earliest sign of mitochondrial dysfunction observed in this study was a significant decrease in the ADP:O ratio for the oxidation of glutamate 1 h post-dosing. At 1.5 and 2 h post-dosing the RCRs for both glutamate- and succinate-supported respiration were significantly decreased. All of the respiratory parameters measured in this study were significantly decreased, with the exception of succinate-supported state 4 respiration which was significantly increased, 2 h after AA administration. Thus, inhibition of mitochondrial respiration preceded overt hepatic necrosis, indicated by an elevation of ALT activity, which was not observed until 3 and 4 h post-dosing. In addition, mitochondrial respiratory dysfunction correlated with morphological alterations. Inhibition of mitochondrial respiration therefore appears to be an early event in the course of AA-induced hepatotoxicity. Cotreatment with NAC (1200 mg/kg) completely prevented the AA-induced impairment of mitochondrial respiration and the development of histopathologic damage. The protection afforded by NAC in these experiments indicates thatN-acetyl-p-benzoquinone imine (NAPQI), the reactive metabolite of AA, is responsible for the observed inhibitory effects, and suggests that mitochondrial dysfunction makes an important, if not essential, contribution to the development of AA-induced hepatotoxicity.

Preclinical toxicology of the anticonvulsant calcium valproate

The oral toxicity of the anticonvulsant calcium valproate with selected comparisons to valproic acid and sodium valproate was evaluated in mice, rats and Beagle dogs. Median lethal doses of the three forms of valproate in rodents ranged from 1100 to 3900 mg/kg. Clinical signs in acute studies and reductions in body weight or body weight gain and food consumption at high doses in rats and dogs during 2-, 13- and 52-week studies were considered to be central nervous system related. In the 13-week study in rats (calcium valproate at 200, 400, 800, 1200 and 1600 mg/kg and sodium valproate at 1200 mg/kg), reduced plasma globulin levels and low white blood cell counts due to suppressed neutrophil maturation were noted at doses of 800 mg/kg and higher. Platelet counts were reduced at 1200 and 1600 mg/kg. Testicular atrophy occurred at 1200 and 1600 mg/kg. In dogs given calcium valproate at 100, 200 and 400 mg/kg for 13 weeks, testicular atrophy was seen at 400 mg/kg and mild hepatocellular changes at all doses. In rats given calcium valproate at 125, 250 and 500 mg/kg for 1 year, reduced plasma protein and globulin levels and a dose-dependent increased incidence and severity of atrophic pancreatitis were noted at 250 and 500 mg/kg. Calcium valproate, given for 1 year to dogs at doses of 50, 100 and 200 mg/kg, was well tolerated. These studies indicated that calcium valproate has a toxicity profile similar to other forms of valproate.

Cheonic Toxicity of the Anticonvulsant Zonisamide in Beagle Dogs

The chronic toxicity of the new anticonvulsant drug zonisamide (1,2-benzisoxazole-3-methanesulfonamide) was evaluated in a detailed 52-week study in which dose levels of 0, 10, 30 and 75 mg/kg/day were administered orally in gelatin capsules to groups of five Beagle dogs per sex. Potential toxicity was based on the effects of zonisamide on body weight and food consumption; clinical and ophthalmic examinations; electrocardiography and heart rates; clinical biochemistry, hematology and urinalysis determinations; organ weights and gross and histopathologic evaluations; electron microscopy of high dose and control male dogs; and plasma zonisamide concentrations. Zonisamide was relatively well tolerated during the study. In animals given 75 mg/kg/day, early body weight losses occurred and therefore, from Weeks 2 and 3 until study termination, for males and females respectively, the high dose was given as two equal portions (i.e., 37.5 mg/kg each) approximately 3-4 hr apart. Clinical laboratory analyses in the dogs given 75 mg/kg revealed a small but statistically significant decrease in plasma albumin concentration and a small increase in alkaline phosphatase activity. In animals given 75 mg/kg, liver weights were increased and a brownish discoloration of the liver was noted grossly at necropsy. No significant light microscopic changes were evident; however, electron microscopic evaluation of the liver tissue from the 5 male dogs given 75 mg/kg revealed the presence of concentric lamellae of paired smooth membranes which were not seen in control animals. At the 10 and 30 mg/kg dose levels, plasma zonisamide concentrations reached steady-state and were proportional to dose, but at 75 mg/kg, plasma levels were disproportionately higher and never achieved steady-state. The results of this study indicated that at the high dose level of 75 mg/kg, chronic administration of zonisamide had a mild effect on the liver, particularly the endoplasmic reticulum.

Preclinical toxicity studies of an adenosine agonist, N-(2,2-diphenylethyl) adenosine

N-(2,2-Diphenylethyl)adenosine (DPEA) has been identified as a potential antipsychotic agent acting via stimulation of adenosine receptors. The projected human therapeutic dose, based on animal studies, is 2-3 mg/kg. DPEA has been tested for potential toxicity in mice, rats, dogs and monkeys. Following single oral doses, median lethal dose values were approximately 10-fold greater in rats than in mice, although similar clinical signs including reduced activity, prostration, and necrosis of the tail were seen in both species. DPEA was well tolerated at daily doses up to 40 mg/kg in rats for 2 weeks. A no observed effect level (NOEL) was not identified in the dog or monkey studies. Reduced activity, dacryorrhea, ptosis, hypothermia, necrosis of the tail, and death occurred in rats given 120 and 160 mg/kg. Pathologic changes consisted of pancreatitis, gastric erosion/ulceration, lymphocyte depletion of the thymus, and pulmonary congestion and hemorrhage at 80 mg/kg or greater. In dogs, sporadic emesis was noted at 12.5 mg/kg and greater, and significant pathologic changes consisted of coronary arteritis associated with myocardial lesions and lymphocyte depletion at 25 and 50 mg/kg, pancreatic acinar necrosis at 50 mg/kg, and renal tubular degeneration at 12.5 mg/kg and greater. Emesis and depression were noted at 25 and 50 mg/kg in monkeys. Renal tubular dilatation and degeneration at 25 and 50 mg/kg were noted in the monkeys. These studies demonstrated that DPEA produced a range of adverse effects in common laboratory animal species.

Hepatotumorigenicity and Peroxisome Proliferation Induced by the Hypolipidemic CI-924 in a Two-Year Study in Male and Female B6C3F1 Mice

The hepatic tumorigenicity of CI-924 (5,5-(1,1-biphenyl)-2,5-diylbis(oxy)(2,2-dimethylpentanoic acid)), a hypolipidemic agent, was evaluated in 50 B6C3F1 mice/sex/dose given drug in the diet at 0, 5, 25, and 75 mg/kg/day for 2 yr. Peroxisomal and drug-metabolizing enzyme determinations, as well as ultrastructural evaluations, were conducted in subsets of these same groups, because drugs of this class cause peroxisome proliferation and hepatic tumors in rodents. CI-924 elicited dose-dependent increases in the incidence of hepatocellular adenomas and carcinomas in both sexes that were statistically significant at 75 mg/kg. Stereologic evaluation revealed significant increases in hepatocellular peroxisome volume ratio, due to increased numbers of peroxisomes, in females at all doses and males at 75 mg/kg. Peroxisomal enzyme activity measurements revealed no change in catalase, but dose-dependent increases in carnitine acetyltransferase and cyanide-insensitive β-oxidation in both sexes. Peroxisome proliferation, determined biochemically or ultrastructurally, was twice as great in females compared to males. Total cytochrome P-450 was increased in both sexes given 75 mg/kg. There were dose-dependent decreases in glutathione S-transferase in males and increased glutathione peroxidase in both sexes at 25 and 75 mg/kg. In conclusion, this study demonstrated that while CI-924 induced hepatic tumors in male and female B6C3F1 mice the associated peroxisome proliferation, while moderate in females, was only weak in the males after 2 yr of exposure.

Renal and hepatic toxicity of a benzopyran-4-one in the Cynomolgus monkey.

The administration of PD 119819, a novel benzopyran-4-one brain dopamine autoreceptor agonist, to Cynomolgus monkeys was followed by deposition of needle-like drug crystals in the bile canaliculi, hepatocytes, proximal renal tubules and renal parenchyma. The crystals were associated with a granulomatous inflammation, and histological and biochemical evidence of hepatic and renal cell damage. Although metabolism differences may be the reason why primates, but not rodents, developed these changes, this form of crystallization appeared to be primarily a result of the insolubility of PD 119189 at alkaline pH.

Interpretation of Clinical Pathology Results in Non-Clinical Toxicology Testing

Abstract Guidelines for interpretation of standard clinical pathology tests and some novel biomarkers in non-clinical toxicology studies are presented. The importance of an integrated approach for clinical pathology interpretation that takes into consideration clinical observations, in-life data, and changes in organ weight and anatomic pathology findings are stressed. Hepatic microsomal enzymes are discussed as test article-effects on microsomal enzymes can have implications for clinical pathology result interpretation. Potential effects of factors unrelated to test article-treatment, including age, gender, genetics, anesthesia, blood collection, diet, fasting, stress, environment, and pregnancy, are discussed. In addition, biological relevance, reporting and use of terminology, statistics, reference intervals, and historical controls are included.

Clinical Pathology in Non-Clinical Toxicology Testing

Abstract Clinical pathology test parameters (hematology, hemostasis, clinical chemistry and urinalysis) generally included for evaluation in non-clinical toxicology studies are identified and their fundamental properties characterized, in order to provide a framework for results interpretation. The importance of designing the clinical pathology component of protocols to optimize the opportunity to achieve study objectives is emphasized as well as the need for inclusion of validated test parameters, the results for which can be interpreted with confidence in the relevant species. Additional tests that may be judiciously included as required are considered: in particular, hormones and acute phase proteins. Microsomal enzymes are discussed as test article-related effects on liver microsomal enzymes can have implications for clinical pathology result interpretation as well as for metabolism and excretion of the test substance and other exogenous and endogenous substances. Features of laboratory novel biomarkers of renal, hepatic, cardiac, and vascular injury are briefly overviewed. In particular, the importance of prudently designing the clinical pathology component of study protocols and integrating clinical pathology findings with other study results in the evaluation of overall safety or risk assessment is stressed.

Early Effects of CI-924 on hepatic peroxisome proliferation, microsomal enzyme induction, PCNA, and apoptosis in B6C3F1 mice and Wistar rats

Abstractu2002The lipid lowering agent 5,5′{[1, 1′-biphenyl]-2,5-diylbis(oxy)}bis[2, 2-dimethylpentanoic acid] (CI-924) is a peroxisome proliferator in rats and mice, but increased the incidence of hepatic tumors in mice only. Male and female B6C3F1 mice and albino Wistar rats were treated with CI-924 at doses ofu20030, 25 and 75 mg/kg for 1, 3, 7 and 28 days. Our aim was to identify species differences potentially related to tumorigenicity and to establish the time course of early events related to or associated with peroxisome proliferation. After 24 h of exposure to CI-924 in the diet there were increases in carnitine acyl transferase and CYP4A1 activity in mice at 25 and 75 mg/kg. In rats, carnitine acyl transferase activity was increased after 24 h and CYP4A1 activity increased after 3 days at 75 mg/kg. Acyl CoA oxidase activity was increased at both doses in male and female rats and mice by 3 days. In general the changes in enzyme activity were of greater magnitude in rats. In contrast to the rapid peroxisome proliferation, increases in the amount ofu2003PCNA were observed in CI-924 treated rats and mice at later times after administration and only at 75 mg/kg. PCNA was increased to a similar extent in both rats and mice, while apoptosis was decreased at both doses of CI-924 after 3 days in female rats, 7 days in male rats, and wasu2003largely unchanged in mice. It was concluded that the sequence of peroxisome proliferation was generally similar in rats and mice. Early changes in cell proliferation and programmed cell death were not directly correlated with subsequent CI-924-induced hepatotumorigenicity.

Absence of correlation between telomerase activity and hepatic neoplasia in B6C3F1 mice.

Telomeres are the physical ends of eukaryotic chromosomes, which maintain chromosome stability and are progressively shortened with aging in somatic cells. The enzyme telomerase elongates telometric DNA and while not usually detectable in human somatic cells is expressed in most human tumors. The present study was conducted to determine if telomerase activity is a marker for spontaneous hepatic neoplastic changes in B6C3F1 mice, a strain frequently used in rodent carcinogenicity studies. Telomerase activity was generally higher in microscopically normal liver tissue from 8-week-old compared to aged mice (110-week-old); however, telomerase activity was not consistently increased in hepatocellular adenomas and carcinomas. It is proposed that, while elevated telomerase activity may modulate human tumor development, modulation of telomerase activity is not a feature of hepatic tumors in B6C3F1 mice and therefore is unlikely to have utility as a molecular marker for hepatic neoplasia in this mouse strain.