It is well known that various kinds of hypolipidemic drugs induce marked changes in the livers of rats and mice. The initial hepatic responses in rodents are marked hepatomegaly, proliferation of peroxisomes in association with changes in peroxisome structure and enzyme composition. Furthermore, since many of hypolipidemic peroxisome proliferators induce hepatocellular carcinomas in both rats and mice, the relationship between peroxisome proliferation and hepatocarcinogenicity of these drugs has become extremely important. However, it has not yet been established whether there are any direct relationships among pharmacological action, peroxisome proliferation and carcinogenicity of these drugs. In order to clarify this task, we have studied the involvement of HGF in hepatocarcinogenesis caused by peroxisome proliferators. After male F-344 rats were orally given Wy-14,643, hepatocarcinomas and (pre) neoplastic nodules were observed in the livers. At that time, the content of HGF and the expression of HGF mRNA were significantly decreased in the liver tumors. These findings may indicate that decreases in hepatic HGF levels are specific events induced by peroxisome proliferators but not by genotoxic carcinogenesis, and that those changes play an important role in the promotion of neoplastic or preneoplastic cell growth induced by peroxisome proliferators. Decrease in HGF induced by peroxisome proliferators such as Wy-14,643 would inhibit the growth of normal hepatocytes and then lend an advantageous circumstance for the selective growth of neoplastic or preneoplastic cells, resulting in the development of growth of tumors.
In general toxicological studies, prothrombin time and activated partial thromboplastin time are routinely measured to assess blood coagulation. Special (problem-driven) tests for blood coagulation are of significance to detect abnormalities and investigate the mechanism of toxicity in detail. In this review, we compiled widely scattered information on blood coagulation testing from different fields in the biological area, and reviewed the methods available and their significance in toxicological studies. The relevant literature cited here reports large species differences in platelet aggregation, coagulation factors or fibrinolysis, and technical limitations. However, the following tests are basically applicable to laboratory animals; (1) assays for individual coagulation factors and protein induced by vitamin K absence or antagonists (PIVKA) to investigate coagulation factor abnormalities; (2) platelet aggregation-, platelet adhesion-, platelet release-tests and von Willebrand factor assay to screen and/or investigate platelet dysfunction; (3) fibrin/fibrinogen degradation products (FDP), D-dimer and thromboelastogram to detect fibrinolitic abnormalities, and assays for plasminogen, plasmin and their activator/inhibitor to investigate fibrinolysis in detail; and (4) bleeding-time to grossly evaluate blood coagulation capability in vivo. An appropriate battery of these tests provides significant information for risk assessment of drugs.
The present communication deals with information regarding the practice of the oral glucose tolerance test and determination of serum fructosamine in laboratory beagles. In the oral glucose tolerance test, a 180-min level was found to be crucial following a gavage administration of 50% glucose solution at 5 mL/kg per body weight under fasting conditions. Serum fructosamine concentration as determined by enzymatic assay ranged between 82 and 123 μmol/L (mean of 104 μmol/L), which was about 0.285 to 0.25 times the value obtained by the chemical method described by Johnson and colleagues. Reasons for differences are ascribed to the presence of substances with reducing potential other than fructosamine in the serum.
The combination of an angiotensin II type 1 receptor blocker (ARB) and a diuretic is effective clinically in treatment of hypertension. As a non-clinical safety evaluation of a combination of the ARB olmesartan medoxomil (OM) and the diuretic hydrochlorothiazide (HCTZ), male and female normotensive rats were administered OM/HCTZ (fixed ratio of 8 : 5) orally by gavage for 26 weeks at dose levels of 0 , 4.88, 16.25, 48.75, 162.5, 487.5, or 1625 mg/kg/day. Additional groups were given 1000 mg/kg/day OM or 625 mg/kg/day HCTZ. Statistically significant and marked decreases in urinary protein excretion were observed in males and females given doses of 16.25 mg/kg/day or higher compared to vehicle-control groups. Increases in blood urinary nitrogen (BUN) were observed in males and females given doses of 16.25 and 162.5 mg/kg/day or higher, respectively. Increased incidence of chronic progressive nephropathy (CPN), a rat-specific spontaneous renal lesion, was observed in males and females given doses of 48.75 mg/kg/day or higher. An additional mechanistic study, consisting of male and female rats given 0, or 162.5 mg/kg/day OM/HCTZ, was conducted to clarify the toxicological significance of the increases in BUN and the increased incidence of CPN described above. This additional study clearly demonstrated that saline-supplementation through free access to saline in the drinking water ameliorated the elevation in BUN and also ameliorated the incidence of CPN. Consequently, the effects on BUN and CPN observed in the first study can be explained by the hemodynamic disturbances caused by the large doses and an exaggerated pharmacological action in volume-depleted normotensive animals. Importantly, the marked decreases in urinary protein were not affected by the saline-supplementation, and indicated that OM/HCTZ elicited a renoprotective effect, probably by an effect on the glomeruli. An additional toxicokinetic study revealed no drug interactions between OM and HCTZ. In conclusion, OM/HCTZ induced a renoprotective effect as well as changes probably attributed to the exaggerated pharmacological action of the ARB with diuretic in normotensive rats. These results suggest that OM/HCTZ may have renoprotective effects in clinical treatment of hypertensive patients.
A variety of catechins exist in green tea and Chinese rhubarb. It is also known that the green tea tannin mixture and its individual tannin components such as (-)-epicatechin 3-O-gallate (ECG) and (-)-epigallocatechin 3-O-gallate (EGCG) suppress renal failure in animals and inhibit the growth of mesangial cells. In addition, gallic acid (GA), a structural constituent of these catechins, induces apoptosis in tumor cell lines. However, the effects of catechins on renal tubular cells have not been investigated. In this experiment, the growth of opossum kidney proximal tubular (OK) cells was inhibited by GA (36.9 ± 9.5%, p<0.01) and EGCG (48.6 ± 16.7%, p<0.01) at 50 μM, and was almost completely inhibited by these compounds at concentrations over 100 μM. Furthermore, ECG inhibited the growth of OK cells at concentrations over 200 μM (52.6 ± 16.5%, p<0.01). The numbers of in situ end-labeled (ISEL) cells in cultures treated with GA at 25 and 50 μM, ECG at 100 and 200 μM, or EGCG at 25 μM were significantly less than those in the cultures treated with high-concentration EGCG (50 μM). In addition, exposure to 50 μM EGCG or 400 μM GA for 24 hr led to a significant increase in fragmented DNA, but ECG did not significantly induce DNA fragmentation compared to the control. These results suggest that EGCG induces mostly apoptosis in OK cells, but the cellular toxicity of GA involves both apoptosis and other mechanisms. Finally, ECG inhibits the growth largely via some mechanism other than apoptosis. This chemical-specific difference of cytotoxic pattern may be dependent on the combination of GA and basal catechin structures, or NADH oxidase (NOX) activity on the OK cell surface.
In fear-conditioning paradigms, rats display a freezing response not only upon presentation of a conditioned stimulus (usually tone), but also when returned to a conditioning chamber (context) in which they received an unconditioned stimulus (usually a foot shock). These paradigms have intrinsic advantages for screening effects of chemicals on learning and memory, although the time-consuming monitoring of freezing time by human observers may be problematic. In this study, an automated apparatus was developed to optimize a fear-conditioning paradigm for screening. We developed an apparatus that records freezing time measured from body movements detected by passive infrared (PIR) sensors. The apparatus detected freezing time as accurately as the human scoring method, and these data were used to determine learning parameters (freezing time to context or to tone) and a non-learning parameter (freezing time to novel context) in the rats. Rats orally administered the neurotoxic compound trimethyltin (TMT) exhibited decreased freezing levels to context, but not to tone or to novel context. These results suggest that this automated method can be an effective part of a screening program.
The administration of certain quinolone antibiotics has been associated with a prolongation of the QT interval on electrocardiogram, and in rare cases ventricular arrhythmias such as torsades de pointes. In this in vivo study using a rabbit arrhythmia model, we assessed the proarrhythmic effects and changes in the QT interval elicited by the administration of NM394 (UFX), an active metabolite of the new quinolone antibiotic prulifloxacin, and three representative quinolones, sparfloxacin (SPFX), gatifloxacin (GFLX) and levofloxacin (LVFX). Chloralose-anesthetized rabbits were co-administered a continuous infusion of methoxamine (15 μg/kg/min) together with NaOH (vehicle, 0.2 mol/L), SPFX (2, 3, 4 mg/kg/min), GFLX (4 mg/kg/min), LVFX (4 mg/kg/min) or UFX (4 mg/kg/min) via the ear vein, and then the effects on electrocardiogram were examined. SPFX and GFLX both prolonged the QT and QTc intervals. GFLX also induced premature ventricular contractions in all 6 rabbits that received it, and subsequently it induced torsades de pointes (TdP) in 3 of the 6 rabbits. SPFX infused at the dose of 4 mg/kg/min induced conduction blocks without inducing TdP, whereas that infused at the lower dose of 3 mg/kg/min induced both conduction blocks and TdP. The infusions with LVFX and UFX did not elicit remarkable prolongations in the QT interval, and none of the animals infused with the agents developed arrhythmia. These findings suggested that LVFX and UFX were less potent than SPFX and GFLX in prolonging the QT interval and inducing life-threatening arrhythmias.
Rice bran glycosphingolipid (RBGSL), one of the glycosphingolipids (GSLs), has been widely used as a food additive, a base of cosmetics, and so on. As a part of the safety assessment of RBGSL, a 13-week repeated dose toxicity study was performed in Wistar Hannover (GALAS) rats. Male and female rats were divided into 4 groups consisting of 8 animals and were given 0, 60, 250, and 1000 mg/kg BW of RBGSL orally 5 times weekly for 13 weeks. During the experiment, no deaths were observed in any groups, and there were no remarkable changes in general appearance, body weight, food and water consumption, hematological and serum biochemical parameters, organ weight and histopathological findings between the control and treated groups. On the basis of these data, the no-observed-adverse effect level (NOAEL) of RBGSL in Wistar Hannover rats was considered to be 1000 mg/kg BW/day or more.