The PIGRET assay, a method for measuring Pig-a gene mutation in reticulocytes, is reliable as a short-term in vivo genotoxicity test: Summary of the MMS/JEMS-collaborative study across 16 laboratories using 24 chemicals.

The in vivo mutation assay using the X-linked phosphatidylinositol glycan class A gene (Pig-a in rodents, PIG-A in humans) is a promising tool for evaluating the mutagenicity of chemicals. Approaches for measuring Pig-a mutant cells have focused on peripheral red blood cells (RBCs) and reticulocytes (RETs) from rodents. The recently developed PIGRET assay is capable of screening >1×106 RETs for Pig-a mutants by concentrating RETs in whole blood prior to flow cytometric analysis. Additionally, due to the characteristics of erythropoiesis, the PIGRET assay can potentially detect increases in Pig-a mutant frequency (MF) sooner after exposure compared with a Pig-a assay targeting total RBCs (RBC Pig-a assay). In order to test the merits and limitations of the PIGRET assay as a short-term genotoxicity test, an interlaboratory trial involving 16 laboratories was organized by the Mammalian Mutagenicity Study Group of the Japanese Environmental Mutagenicity Society (MMS/JEMS). First, the technical proficiency of the laboratories and transferability of the assay were confirmed by performing both the PIGRET and RBC Pig-a assays on rats treated with single doses of N-nitroso-N-ethylurea. Next, the collaborating laboratories used the PIGRET and RBC Pig-a assays to assess the mutagenicity of a total of 24 chemicals in rats, using a single treatment design and mutant analysis at 1, 2, and 4 weeks after the treatment. Thirteen chemicals produced positive responses in the PIGRET assay; three of these chemicals were not detected in the RBC Pig-a assay. Twelve chemicals induced an increase in RET Pig-a MF beginning 1 week after dosing, while only 3 chemicals positive for RBC Pig-a MF produced positive responses 1 week after dosing. Based on these results, we conclude that the PIGRET assay is useful as a short-term test for in vivo mutation using a single-dose protocol.

Measuring reproducibility of dose response data for the Pig-a assay using covariate benchmark dose analysis.

The reproducibility of the in vivo Pig-a gene mutation test system was assessed across 13 different Japanese laboratories. In each laboratory rats were exposed to the same dosing regimen of N-nitroso-N-ethylurea (ENU), and red blood cells (RBCs) and reticulocytes (RETs) were collected for mutant phenotypic analysis using flow cytometry. Mutant frequency dose response data were analysed using the PROAST benchmark dose (BMD) statistical package. Laboratory was used as a covariate during the analysis to allow all dose responses to be analysed at the same time, with conserved shape parameters. This approach has recently been shown to increase the precision of the BMD analysis, as well as providing a measure of equipotency. This measure of equipotency was used here to demonstrate a reasonable level of interlaboratory reproducibility. Increased reproducibility could have been achieved by increasing the number of cells scored, as this would reduce the number of zero values within the mutant frequency data. Overall, the interlaboratory trial was successful, and these findings support the transferability of the in vivo Pig-a gene mutation assay.

Evaluation of the RBC Pig-a assay and the PIGRET assay using benzo[a]pyrene in rats.

The red blood cell (RBC) Pig-a assay has the potential to detect the in vivo mutagenicity of chemicals. Recently, use of the Pig-a assay with reticulocytes (the PIGRET assay) reportedly enabled the in vivo mutagenicity of chemicals to be detected earlier than using the RBC Pig-a assay. To evaluate whether the PIGRET assay is useful and effective as a short-term test, compared with the RBC Pig-a assay, we performed both assays using benzo[a]pyrene (BP), which is a well-known mutagen. BP was used to dose 8-week-old male rats orally at 0, 75.0, 150, and 300mg/kg administered as a single administration. Peripheral blood samples were then collected on days 0, 7, 14, and 28 after treatment and were used in both assays. In the treatment groups receiving 150mg/kg of BP or more, both the RBC Pig-a assay and the PIGRET assay detected the in vivo mutagenicity of BP. In the 300mg/kg treatment group, in which a significant increase in the mutant frequency (MF) was observed at all the sampling points using both the RBC Pig-a assay and the PIGRET assay, the reticulocyte (RET) Pig-a MF was higher than the RBC Pig-a MF on days 7 and 14 after treatment; nevertheless, the negative control RET Pig-a MF was comparable to the negative control RBC Pig-a MF. In addition, the RET Pig-a MF began to increase after day 7 and reached a maximum value on day 14 after treatment, whereas the RBC Pig-a MF increased continuously from day 7 until day 28 after treatment. These results indicate that the PIGRET assay has a higher sensitivity than the RBC Pig-a assay and that the PIGRET assay is useful for the earlier detection of the in vivo mutagenicity of chemicals, compared with the RBC Pig-a assay.

Effects of telmisartan on right ventricular remodeling induced by monocrotaline in rats.

The present study investigated whether telmisartan, an angiotensin II type 1 receptor antagonist, has cardioprotective effects on monocrotaline-induced right ventricular (RV) remodeling in rats. Six-week-old male Wistar rats were divided into control group (CONT), monocrotaline (60 mg/kg, i.p.)-treated group (MCT), monocrotaline (60 mg/kg, i.p.) + telmisartan (3 mg/kg per day, p.o.)-treated group (MCT+TEL), and telmisartan (3 mg/kg per day, p.o.) alone-treated group (TEL). Hearts were excised after echocardiography examinations at day 25. Significant increase in RV weight and histologically remarkable fibrosis in RV sections were observed in MCT. Tricuspid annular plane systolic excursion, a parameter for RV systolic function, significantly decreased in MCT. These RV hypertrophy, fibrosis, and dysfunction were inhibited in MCT+TEL. In MCT, the acceleration time/ejection time ratio of pulmonary artery flow velocity, an index of pulmonary hypertension, significantly decreased. This decrease was not affected in MCT+TEL. In MCT, expressions and activities of matrix metalloproteinase (MMP)-2 and MMP-9, which play a critical role in cardiac remodeling, significantly increased in the RV. In MCT+TEL, these increases in expressions and activities were inhibited. MCT showed about 2-fold increase in transforming growth factor-beta1 expression compared with CONT, and such an increase was not decreased in MCT+TEL. There were no significant changes of these parameters in TEL compared with CONT. These results suggest that telmisartan could attenuate the monocrotaline-induced RV remodeling through improvements of RV hypertrophy, fibrosis, dysfunction, and inhibition of MMPs.

Captopril attenuates matrix metalloproteinase-2 and -9 in monocrotaline-induced right ventricular hypertrophy in rats.

Little is known about the influence of angiotensin converting enzyme (ACE) inhibitors on matrix metalloproteinase (MMP) in right ventricular remodeling. We investigated the effect of captopril, an ACE inhibitor, on MMP-2 and MMP-9 in monocrotaline-induced right ventricular hypertrophy. Six-week-old male Wistar rats were injected intraperitoneally with monocrotaline (60 mg/kg) or saline. The rats were administrated captopril (30 mg/kg per day) or a vehicle orally for 24 days from the day of monocrotaline injection. At day 25, echocardiography was performed and hearts were excised. Expressions and activities of MMP-2 and MMP-9 were measured by Western blotting and by gelatin zymography, respectively. In monocrotaline-injected rats, right ventricular weight/tail length ratio increased significantly. Histological analysis revealed cardiomyocyte hypertrophy and fibrosis in right ventricular sections. Echocardiography showed right ventricular dysfunction compared with saline-injected rats. The right ventricular hypertrophy, fibrosis, and dysfunction were inhibited by captopril. However, captopril did not attenuate an increase in pulmonary artery pressure. MMP-2 and MMP-9 expressions and activities in right ventricles increased significantly in monocrotaline-injected rats and captopril inhibited them. These findings indicate that captopril attenuates the development of monocrotaline-induced right ventricular hypertrophy in association with inhibition of MMP-2 and MMP-9 in rats.