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Carcinogenic potential of the thiazolidinedione antidiabetic troglitazone was assessed in 104-week studies in mice and rats. Mice were given 50, 400, or 800 mg/kg, male rats 100, 400, or 800 mg/kg, and female rats 25, 50, or 200 mg/kg. Vehicle and placebo controls were included. Survival was significantly decreased in both sexes of both species at high doses, but was adequate for valid evaluation of carcinogenicity. Hypertrophy and hyperplasia of brown adipose tissue was observed in both species at all doses, and fatty change and hypocellularity of bone marrow was noted in mice at all doses and in female rats at 50 and 200 mg/kg. Hepatocellular vacuolation was observed in mice at 400 and 800 mg/kg, and centrilobular hepatocellular hypertrophy occurred in rats at > or = 200 mg/kg. Ventricular dilatation, myocardial fibrosis, and atrial myocyte karyomegaly in male rats at 400 and 800 mg/kg and female rats at all doses were morphologically similar to spontaneous lesions, but incidence and severity were increased compared with controls. In mice, the incidence of hemangiosarcoma was increased in females at 400 mg/kg and in both sexes at 800 mg/kg. The incidence of hepatocellular carcinoma was increased in female mice at 800 mg/kg. Troglitazone exposure [AUC((0-24))] at the lowest dose associated with increased tumor incidence in mice was 16 times human therapeutic exposure at 400 mg daily. No tumors of any type were increased in rats at exposures up to 47 times therapeutic exposure.

Aims-The two electron reduction of quinones to hydroquinones by NAD(P)H quinone oxidoreductase (NQO1) plays an important role in both activation and detoxification of quinone and similarly reactive compounds. A single nucleotide polymorphism at exon 6 leads to an amino acid change at codon 187 from proline to serine. The variant allele has been associated with decreased NQO1 enzyme activity and increased cancer risks. The aim of this study was to develop a rapid genotyping procedure for epidemiological and clinical research into the potential biological and toxicological implications associated with this genetic polymorphism. Methods-A high throughput genotyping method using fluorogenic probes has been developed to screen this single nucleotide polymorphism. This assay utilises the 5' nuclease activity of Taq polymerase in conjunction with fluorogenic TaqMan probes. The TaqMan genotyping procedure was validated by a restriction fragment length polymorphism method and direct sequencing. Results-This method can be used for the rapid screening of known polymorphisms in large populations. In a population of 143 unrelated individuals, Pro/Pro (wildtype), Pro/Ser (heterozygous), and Ser/ Ser (mutant) genotypes were 69.2%, 26.6%, and 4.2%, respectively. Conclusions-This genotyping method is highly accurate and could be applied to automated large scale genotyping studies. (J Clin Pathol: Mol Pathol 1999;52:295-299) Keywords: NAD(P)H quinone oxidoreductase; genotyping; TaqMan NAD(P)H quinone oxidoreductase (NQO1; EC1.6.99.2; DT diaphorase) is a cytosolic flavin containing enzyme that catalyses the two electron reduction of quinone substrates.

Thiazolidinediones (TZDs) are effective for the treatment of adult-onset insulin-resistant diabetes. Unfortunately, TZDs are associated with sporadic hepatic dysfunction that is not predictable from experimental animal studies. We investigated the response of isolated rat and human hepatocytes to various TZDs using biochemical assays, coherent multiprobe fluorescence microscopy and flow cytometric analyses. The results identified direct effects of TZD on mitochondria from live human and rodent hepatocytes. The multiprobe fluorescence assays showed disruption of mitochondrial activity as an initiating event followed by increased membrane permeability, calcium influx and nuclear condensation. Other TZD-related cellular effects were increased hepatic enzyme leakage, decreased reductive metabolism and cytoplasmic adenosine triphosphate depletion. Mitochondrial effects were similar in cryopreserved hepatocytes from diabetic or non-diabetic donors. Peripheral blood mononuclear cells (PBMCs) had baseline mitochondrial energetics and metabolism comparable with isolated hepatocytes. Mitochondrial effects in isolated hepatocytes were found in human PBMCs exposed to the TZDs. The relative potency of TZDs for causing hepatocyte and PBMC effects was troglitazone > pioglitazone > rosiglitazone. These studies clearly demonstrated that hepatic alterations in vitro are characteristic of TZDs, with only quantitative differences in subcellular organelle dysfunction. Monitoring mitochondrial function in isolated PBMCs may be beneficial in diabetics undergoing TZD therapy.

The calcium salt of valproic acid (Valontin) has been proposed for use in the treatment of absence, myoclonic, and tonic clonic seizures of the primarily generalized type. The present study was conducted to determine the teratogenic potential of calcium valproate in rabbits. Groups of 20 Dutch-belted rabbits were given oral doses of 50, 150, or 350 mg/kg on days 6-18 of gestation. A reference group was given 350 mg/kg sodium valproate and control groups were untreated or given vehicle alone. Animals were observed daily and body weights were recorded on gestation days 0, 6, 13, 18, and 30. Litter and fetal parameters were evaluated following uterotomies on day 30. No drug-related clinical signs or deaths occurred. Postimplantation loss and the incidence of malformed vertebrae and ribs, rudimentary or absent pollices, and extra vertebrae and ribs were increased at 350 mg/kg with both calcium and sodium salts of valproic acid. At the 150-mg/kg dose level, calcium valproate markedly increased the incidence of supernumerary ribs. No teratogenic or embryotoxic effects were seen with calcium valproate at 50 mg/kg. These data indicate that the sodium and calcium salts of valproic acid exhibit teratogenic potential in rabbits.