The single cell gel electrophoresis (comet) assay is a simple and effective method for detecting DNA damage in cells with or without the capability of cell division. Methyl methanesulfonate (MMS), as a genotoxic compound that reacts with DNA directly, was confirmed for its DNA damage potential by in vivo comet assay in multiple organs such as liver, kidneys and bone marrow in mice and acetaminophen (APAP), a widely used analgesic drug, was evaluated for whether it possesses DNA damage potential or not. Furthermore, cytotoxicity was verified by hematology and /or blood chemistry simultaneously. Male Crj:CD1(ICR) mice were intraperitoneally once treated with MMS at 50, 100, and 150 mg/kg, and APAP at 12, 60, and 300 mg/kg. These organs were collected at 4 and 24 hr after treatment, and the comet assay was performed concomitantly with hematology and/or blood chemistry. The results showed that MMS induced a significant concentration-dependent increase in the frequency of tailed nuclei (DNA damage), tail moment, % DNA in the tail, and tail length in the liver, kidneys and bone marrow at both time points. With regard to hematology and blood chemistry results, nephrotoxic markers were not changed, but aspartate aminotransferase (AST) and alanine aminotransferase (ALT) increased in the 150 mg/kg-treated group, and bone marrow counts (BMC) decreased in all of the treatment groups 24 hr after treatment. These results suggested that DNA damage observed in the kidneys was due to genotoxicity, not nephrotoxicity. The DNA damage was more severe at 4 hr than 24 hr after treatment. This might indicate that the decrease in DNA damage was due to detoxification, repair of the lesions induced by the treatment, or cell turnover, all of which would reduce cellular damage. On the other hand, APAP induced increases in plasma AST and ALT levels in the highest dose group only, and the DNA damage in the liver increased at the same dose. These results suggest that the in vivo comet assay might be used to detect the DNA damage induced by MMS and the subsequent DNA repair in mouse liver, kidneys and bone marrow. APAP at the highest dose induces DNA damage in liver. Blood chemical results may indicate that the DNA damage by APAP treatment was attributable to hepato-cytotoxicity, because DNA damage and hepato-cytotoxicity were detected at the same doses.
Plasma protein binding is an important factor for the kinetics of drugs and how they act since it is the first step in drug distribution. Physiological changes in pregnancy, which include plasma composition, can affect drug binding and subsequent drug response. In the present study, we investigated the toxicokinetics (TK) and/or toxicodynamics (TD) of diclofenac and propranolol comparing pregnant Sparague-Dawley (SD) rats with non-pregnant SD rats in terms of protein binding and drug distribution. Diclofenac and propranolol are reported to bind to albumin and α1-acid glycoprotein (AGP), respectively. After a single administration of diclofenac, the area under plasma concentration-time curve (AUC) based on free diclofenac in pregnant rats was 3.9 times higher than that in non-pregnant rats. This difference is considered to be due to a lower concentration of serum albumin and a higher concentration of non-esterified fatty acid (NEFA) that inhibits drug binding to albumin, in pregnant rats. In histopathological examination, more severe gastrointestinal toxicity was observed in pregnant rats at 24 hr after dosing. This severe toxicity was likely to be correlated with the higher AUC. With respect to propranolol, the difference of the AUC based on free propranolol was not clear although the concentration of serum AGP was lower in pregnant rats. However, the binding analysis data suggested a difference of protein binding at a lower propranolol concentration range. Consequently, lowered serum proteins and increased NEFA in pregnant rats can lead to low protein binding, subsequent increase in free drug concentrations, and eventual increase in the TD of drugs.
This study comprehensively describes the effects of various levels of food reduction on a wide range of toxicological parameters in dietary-optimized rats (fed with approximately 75% of ad libitum food consumption daily; 16 g and 22 g/day for females and males, respectively) that has been established as a nutritionally appropriate and well-controlled animal model in conducting toxicity studies. Toxicological parameters, including general condition, ophthalmology, clinical pathology and anatomic pathology, were examined in dietary-optimized Crl:CD(SD) female and male rats fed 16 g and 22 g/day (control), 12 g and 17 g/day (75% group), 8 g and 11 g/day (50% group), or 4 g and 6 g/day (25% group), respectively for 2 weeks. There was mortality and morbidity including reddish urine in 25% group females. The reddish urine was identified as “hemoglobinuria” that resulted from extra/intra-vascular hemolysis induced by severe food reduction. Hemoconcentration, decreased leukocytes and platelets, decreases in nutritional elements (serum glucose, protein, and lipids), increased aspartate aminotransferase and alanine aminotransferase, imbalanced electrolytes, and/or decreased urinary pH were observed in all restriction groups. Histopathologically remarkable changes included erythrophagocytosis in the spleen/liver and renal tubular necrosis with hyaline cast/droplets in 25% group; in addition to bone marrow depletion, lymphoid depletion in thymus/spleen/lymph node, and/or decreased secretion in the prostate/seminal vesicle in all restriction groups. Most of these changes were considered attributable to nutritional deficiency, dehydration, accelerated protein catabolism, stress and/or hemolysis secondary to severe food reduction. These results will enable toxicologists to help distinguish primary drug-induced effects from secondary changes associated with decreases in food consumption.
Serum acetaminophen concentrations are of critical importance in determining the need for acetylcysteine therapy after acute acetaminophen overdose. Limited data suggest opioid co-ingestion might alter acetaminophen pharmacokinetics. The present study was designed to examine serum acetaminophen concentrations after acute overdose, and to compare between patients that co-ingested an opioid and those that did not. A prospective study of consecutive patients that presented to hospital within 16 hr of acute acetaminophen overdose. Equivalent 4-hr acetaminophen concentrations were calculated using the serum acetaminophen concentration at a fixed interval 3 to 16 hr after ingestion. Groups were compared using Mann Whitney tests. There were 990 patients; 295 (29.8%) had co-ingested an opioid, and 695 had not. The median (interquartile range) stated dose was 10 g (6-16 g) vs. 10 g (7-16 g) respectively (P = 0.94), interval between ingestion and acetaminophen determination was 4.5 hr (4.0-6.0 hr) vs. 4.5 hr (4.0-5.5 hr) respectively (P = 0.41), and serum acetaminophen concentration was 56 mg/l (24-105 mg/l) va. 60 mg/l (23-129 mg/l) respectively (P = 0.25). A positive relationship was noted between stated dose and equivalent 4-hr serum acetaminophen concentration, but did not differ between groups. The acetaminophen dose-concentration relationship was similar in patients that did and did not co-ingest an opioid. Therefore, early serum acetaminophen concentrations can be used to determine the extent of drug exposure, irrespective of whether an opioid has been co-ingested.
Depression is one of the frequently-observed psychiatric symptoms associated with nicotine (NC) use. In the present study, considering the unique effects of NC (e.g. antidepressant effects have also been reported), the time course of the NC-induced depressive behavioral alterations in a mouse model was compared with a typical depression-inducing stressor. Furthermore, based on the involvement of cannabinoid (CB) receptors in the behavioral effects of NC, the effects of antidepressants including CB ligands (CBs) against the NC-induced behavioral alterations were also investigated. Repeated subcutaneous NC treatments (0.3 mg/kg, 4 days), like repeated immobilization stress (IM) treatments (10 min, 4 days), caused prolonged depressive effects (increased immobility time) at both 2 hr and 1 day time points after the last treatment in the tail suspension test. However, in the NC group, depressive effects (suppressed swimming behaviors) were observed only at the 2 hr time point in the forced swimming test. The antidepressants amitriptyline, clomipramine and fluvoxamine, the endogenous mixed CB agonist/antagonist virodhamine and the anandamide-like cannabimimetic O-2093 provided antagonistic effects against the depressive behaviors in the tail suspension test. However, in the forced swimming test, NC-induced depressive behaviors were antagonized only by the CBs virodhamine and O-2093. The present results demonstrated depressive effects of NC in two typical behavioral tests, which support the risk of repeated NC use. The shortened behavioral alterations in the forced swimming test, as compared to the IM group, seemed to reflect the neuronal modifications peculiar to NC, which are antagonized by some CBs.
A simple, sensitive, rapid and specific enzyme linked immunosorbent assay (ELISA) for quantitative measurement of aflatoxin B1 (AFB1) in urine samples was developed in this study. Polyclonal antibodies were raised against a C1-carboxymethyl oxime (CMO) derivative of AFB1 conjugated bovine serum albumin (BSA). AFB1-C8-penicillinase (AFB1-C8-P) and AFB1-C1-carboxymethyl oxime-penicillinase (AFB1-CMO-P) were prepared and used as tracer molecule. A heterologous combination of antibody and enzyme conjugates (AFB1-C1-CMO-BSA and AFB1-C8-P) proved to work better with respect to specificity and sensitivity. Ig purified antibody (4 µg/well) was coated onto the pre-coated (BSA) wells of microtiter plate. The assay procedure was completed within 3 hr and the sensitivity was calculated to be from 200 pg/ml. The standard curve was linear up to 10 ng/ml so was able to detect high concentration of AFB1 in sample. Affinities were calculated for homologous and heterologous system in which the heterologous system showed better affinities (1.9 × 108 M-1). The antibody prepared in this study showed minimal cross-reaction with structurally related molecules being affected by homology and heterology of the assay system that is the site of conjugation of carrier protein for antibody production using the hapten BSA conjugate and the site of enzyme conjugated on the hapten molecule used as tracer as well as direct and indirect coating of antibody on the surface of microtiter plat. The results reported here indicated that the heterologous combination of antibody and enzyme conjugate performs better in assay qualities in general. More than 90% recovery of AFB1 added to stripped urine samples were observed in this type of assay. Inter and intra-assay percent of coefficient of variations for ten successive assays were found to be 10.2 and 6.9% respectively. Logit -log transformation of standard curve and sample dilution with urine sample containing no AFB1 in a serial manner exhibited parallel line with the slope of −1.03 and −1.03 respectively. A correlation of 0.90 was found between the ELISA reported in this study and radioimmunoassay (RIA) of AFB1 in urine samples.
The aim of this study is to investigate the effect of the pharmacokinetic profile of tacrolimus on its pancreatic toxicity and efficacy in rats. For toxicity evaluation, doses of 0.03, 0.1, or 0.3 mg/kg/day were given once daily for 8 days in the bolus intravenous injection groups. In the continuous intravenous infusion groups, tacrolimus was infused using an Alzet® osmotic mini-pump for 9 days at the same doses. Pancreatic insulin content decreased dose-dependently in both the bolus intravenous injection and continuous intravenous infusion groups, and there was no significant difference between the decreases caused by the two dosing regimens. At 0.03 mg/kg, continuous intravenous infusion did not cause glucose intolerance, but bolus intravenous injection induced significant and dose-dependent glucose intolerance. The pharmacokinetic data indicated that continuous intravenous infusion resulted in a sustained blood drug concentration with an area under the curve (AUC) similar to that obtained with the bolus administration at the same dose. For efficacy evaluation, donor ear grafts were transplanted to the lateral thoraxes of recipients. Tacrolimus doses of 0.01, 0.1, or 1 mg/kg/day were administered from day 0 to day 13. Both bolus intramuscular administration and continuous intravenous infusion prolonged skin allograft survival dose-dependently, and there was no significant difference between the median survival times of groups given the same doses. To summarize, the sustained-release of tacrolimus resulted in a steady blood drug concentration with an AUC similar to that of the bolus administration. In rats, it was better tolerated and just as efficacious as the bolus administration without producing a higher maximal blood concentration (Cmax). These results indicate that the sustained-release formulation has the potential to improve the safety of tacrolimus.
Embryonic mortality and intrauterine growth retardation (IUGR) are induced by exposure of rodents to xenobiotic agents during the pregastrulation period of development. We examined the time course of the effects of methyl methanesulfonate (MMS), an alkylating agent, on conceptus development in order to clarify the relative roles of the embryo and the placenta in their induction. Pregnant rats were treated orally with a single dose of MMS (200 mg/kg) in the morning of gestation day (GD) 6 (peri-implantation stage). Embryonic mortality was increased on GD12 and thereafter by MMS treatment, with newly dead embryos showing placental hypoplasia at GD12. Embryo or fetal weight was also smaller for MMS-treated dams than for control dams from GD14 to GD20. The labyrinth zone and junctional zone (JZ) of the placenta were thinner in MMS-treated rats from GD12 to GD17 and from GD12 to GD20 (except for GD17), respectively. Furthermore, MMS-treated dams showed a smaller number of glycogen cells in the JZ on GD14. In contrast, the placental glycogen concentration was higher and the expression of glucose transporter 1 in the JZ remained at GD20. These results indicate that exposure of pregnant rats to MMS at the peri-implantation stage of embryogenesis affects placental development and growth. The placental impairment induced by MMS was likely responsible for the embryonic death observed 6 days after exposure of dams to this agent as well as for the IUGR of surviving embryos or fetuses throughout the gestation period.
Most patients with hepatic porphyria exhibit neuropsychiatric symptoms, including abdominal pain, peripheral neuropathy, confusion, insomnia and mental disturbances such as anxiety and depression. Although heme deficiency and accumulation of heme precursors are thought to be responsible for neuropsychiatric manifestations in patients with acute porphyria, the pathogenetic mechanisms remain poorly understood. In the present study, we observed psychiatric behaviors in mice with hepatic porphyria induced by the ingestion of a griseofulvin (GF)-containing diet over a period of 12 weeks. GF ingestion by the mice caused an accumulation of porphyrins in the feces and a decrease in heme in the liver; these effects were observed throughout the entire duration of the experiment, with maximum levels observed after circa 1 week of ingestion of this diet. In addition, the mice developed enlargement of the liver, hepatocyte injury, and cholestasis. Mice with hepatic porphyria manifested an anxiety-like behavior by the long-term treatment (over 5 weeks) in a GF-dose and duration dependent manner. The hepatic porphyria mice also manifested depression-like behaviors by the short-term treatment (3 weeks) of GF2.0, which was reversed by administration of anti-depressant, imipramine. In conclusion, this study for the first time demonstrated psychiatric manifestations in GF-induced hepatic porphyria mice. The present results suggest that model animals could be useful for elucidating the mechanisms underlying psychiatric manifestations in syndromes such as hepatic porphyria and hepatic encephalopathy that are associated with the impairment of hepatic function.
The influences of inhaling particulate air-pollutants on hematopoiesis and myocardial oxidative stress were investigated in mice by intratracheal instillation (IT) of diesel exhaust particles (DEP), its dichloromethane soluble-component (DMSC) or residual particle-component (RPC). After IT, time courses of cytokine levels in bronchial alveolar lavage fluid (BALF), peripheral blood cell count, myocardial myeloperoxidase (MPO) activity and myocardial chemokine levels were observed for 24 hr. RPC caused sustained blood neutrophilia while that caused by DEP and DMSC was transient. RPC also caused sustained elevations of granulocyte colony-stimulating factor (G-CSF) and interleukin (IL)-6 levels in BALF. Furthermore, IL-1ß level in BALF in the RPC group was significantly elevated at 24 hr after IT. Significant positive correlations were observed between blood neutrophil count and IL-6/G-CSF levels in BALF. MPO activity in the myocardium was increased by RPC at 12 and 24 hr after IT while the activities in the kidney and the liver were not affected. Significant correlation was also observed between myocardial MPO activity and blood neutrophil count at 12 hr after IT, for all three substances. From these results, it was concluded that particle component of DEP may enhance myocardial oxidative stress via blood neutrophilia and the elevation of cytokine levels in BALF.
Reactions of nitrofuran antibiotics (nitrofurazone (NFZ) and frazolidone (FZD)) with hypochlorite in aqueous solution were investigated under the conditions that simulate wastewater disinfection. The chlorination byproducts were determined by high performance liquid chromatography. At the levels of 5 μM, NFZ reacted rapidly with free chlorine in neutral pH (7.0), while the FZD-hypochlorite reaction was reasonably slow under the same pH. Nevertheless, the strong mutagenic parents disappeared completely after the hypochlorite reactions, and the chlorination byproducts were observed to exert a weak mutagenic effect on Salmonella typhimurium TA100 without S9-mix. The extent of the reactions depended on the chlorine dose, solution pH and compound structures.
The aim of this study was to assess the cardiovascular effect of MA-2029, a selective motilin receptor antagonist highly expected for the treatment of irritable bowel syndrome (IBS). MA-2029 inhibited the human ether-a-go-go-related gene (hERG) current at 100 µg/ml, but shortened action potential duration (APD) in isolated guinea pig papillary muscles at 10 and 100 µg/ml and the corrected QT (QTc) interval after oral administration of 30 and 300 mg/kg in conscious telemetered dogs. The discrepancy was probably caused by blockade of the Ca2+ channel because MA-2029 inhibited the Ca2+ current in isolated guinea pig myocytes. MA-2029 at 100 µg/ml also decreased the maximum rising velocity and action potential amplitude in the action potential study, indicating that MA-2029 has Na+ channel blocking potential. In the cardiovascular study, MA-2029 at 30 mg/kg induced slight cardiovascular changes such as hypotension, QTc shortening, and PR prolongation possibly caused by Ca2+ channel blockade. The plasma concentration at 4 hr after 30 mg/kg administration was 2.10 µg/ml, 200-fold higher than the effective concentration of MA-2029 as a motilin receptor antagonist. These results suggest that MA-2029 has sufficient cardiovascular safety although it inhibits multiple ion channels at supra-effective concentrations. On the other hand, cisapride, an effective IBS drug, showed clear hERG inhibition and APD prolongation at 100 ng/ml. Cisapride exhibited a narrow safety margin because it caused QT prolongation potential even at the therapeutic concentration. In conclusion, MA-2029 is a novel drug highly expected for the treatment of IBS with lower cardiovascular risk than cisapride.
Dietary rapeseed (canola) oil (CO) given as the only fat nutrient shortens life in stroke-prone spontaneously hypertensive rats (SHRSP), compared with SHRSP given soybean oil (SO) instead of CO. CO ingestion increases plasma lipids and causes renal lesions in SHRSP and in spontaneously hypertensive rats (SHR), and increases plasma lipids also in Wistar Kyoto (WKY) rats, a normotensive counterpart of SHR. This study examined whether or not such unfavorable effects of CO are restricted to these closely related strains. For this purpose Wistar rats, the strain from which these strains were derived, were fed a diet containing 10% CO or SO as the sole fat nutrient for 10 weeks, and changes in clinical signs, urinalysis, blood biochemistry and pathology were compared. CO ingestion did not induce any abnormalities in Wistar rats, except significant increases in plasma concentrations of aldosterone and Na+, compared with the SO group. Thus, the unfavorable effects of CO ingestion appear to be restricted to SHRSP and its closely related strains. The role of increased aldosterone and Na+ in the unfavorable events caused by CO in SHRSP, SHR and WKY rats, and any factors which could induce such increases in aldosterone and Na+, remain to be elucidated.
Morinda citrifolia (noni) fruit juice use has increased greatly within the past decade, with more than 80,000,000 liters being consumed world wide. With increasing widespread use and the potential use among pregnant women, a prenatal developmental toxicity test was conducted to further evaluate the safety of noni juice. Freeze-dried noni fruit puree from French Polynesia was administered daily by gastric intubation to separate dose groups (n = 12) of pregnant Sprague Dawley rats at 1.72, 3.43, and 6.86 g/kg body weight, with a control group receiving water in place of noni. The dose schedule was followed from the first day of gestation until one day prior to expected delivery, 21 days. There were no symptoms of toxicity in the pregnant dams. There was no difference between the control and any noni group in the number of live fetuses, resorptions, fetal weight and length, or skeletal abnormalities. No dead fetuses, gross external malformations, or internal organ defects were observed in any group. These findings do not indicate that toxicity from noni juice to developing embryos and fetuses is expected.
In order to elucidate the involvement of metallothionein (MT) in radiation carcinogenesis, we examined the susceptibility of MT-I/II null mice to carcinogenesis and oxidative DNA damage resulting from X-irradiation. Eight-week-old female MT-I/II null mice and wild-type mice were exposed to whole-body X-irradiation at doses of 1.0, 1.5 or 2.0 Gy once a week for 6 weeks. Incidence of thymic lymphoma was determined at 24 weeks after the first exposure to X-irradiation. The frequency of thymic lymphomas induced by X-irradiation (at 1.5 and 2.0 Gy) was significantly higher in MT-I/II null mice than in wild-type mice. In addition, although the levels of 8-hydroxy-2'-deoxyguanosine (8-OHdG) were increased in the serum and urine of both strains of mice 24 hr after exposure to a single bout of whole body X-irradiation, these increases were significantly greater in the MT-I/II null mice than in the wild-type mice. Thus, the present results suggest that MT plays a protective role against carcinogenesis and oxidative DNA damage caused by X-irradiation.
Myocardial necrosis is a serious adverse effect that results from the administration of some medications; therefore, when it is observed during preclinical studies it becomes a major drug development concern. Although data from preclinical monkey studies are generally extrapolated to predict effects in humans, few reports have described any mechanism that might explain the occurrence of myocardial necrosis. For this reason, we examined the association between hypokalemia and myocardial necrosis in monkeys. Four female cynomolgus monkeys (Macaca fascicularis) were treated with 50 mg/kg/day hydrochlorothiazide (a thiazide diuretic used for antihypertensive therapy) for 1 or 2 weeks. Clinical, hematological, plasma biochemical, and pathological examinations were conducted. Two animals were kept in a hypokalemic state from day 3 of dosing on, and their mean plasma potassium levels were 2.52 ± 0.24 and 2.60 ± 0.24 mmol/l. These animals were necropsied after 1 week of dosing due to an aggravated general condition. A flattened T-wave was noted during electrocardiography. A transient increase in plasma cardiac-specific troponin-I and multifocal myocardial necrosis also occurred. The rest of the animals were occasionally hypokalemic, with mean plasma potassium levels of 3.13 ± 0.31 or 2.96 ± 0.30 mmol/l. These animals were necropsied after 2 weeks of dosing. One animal showed evidence of focal myocardial necrosis and a transient increase in plasma cardiac-specific troponin-I. These data suggest that the severe hypokalemia induced by hydrochlorothiazide is likely to be associated with myocardial necrosis in monkeys.