N-Nitrosocompounds, which induce cancers in various organs, may be formed endogenously with intake of amino compounds such as secondary amines and sodium nitrite (NaNO2) in combination. The present study was performed to investigate whether three amino compounds, 1-methyl-9H-pyrido[3,4-b]indole (harman), 9H-pyrido[3,4-b]indole (norharman) and 2-amino-1,3,4-triazole (amitrole), might be converted in vivo to compounds capable of promoting hepatocarcinogenesis when given with NaNO2. However, in an 8-week model, no modifying potential was evident in terms of numbers and areas of putative preneoplastic glutathione S-transferase placental form (GST-P)-positive foci in any of the groups receiving paired treatments. These results demonstrate that combinations of harman, norharman and amitrole with NaNO2 lack promoting effects for liver carcinogenesis in our medium-term bioassay system.
Diethylstilbestrol (DES) was administered subcutaneously at 0.5, 1.5 or 4.5 μg/kg/day (DES 0.5, 1.5 and 4.5 groups, respectively) to pregnant SD rats daily on days 7-21 of gestation, to investigate its effects on the development and functions of the reproductive system in their male offspring. Of the 10 pregnant rats in the DES 4.5 group, only 1 delivered, and this rat could not suckle the pups. Rat pups in the DES 0.5 and 1.5 groups were autopsied at 1, 3, 6 and 15 weeks after birth. The testosterone concentrations in the DES 1.5 and 0.5 groups at 6 weeks were significantly decreased and the plasma LH concentrations were not altered. In the DES 1.5 group, DES treatment did not change the volume of the sexually dimorphic nucleus in the preoptic area (SDN-POA) in the male offspring, although this dose of DES increased the volume of SDN-POA in female offspring. The DES treatment altered frequencies in the cycles of the seminiferous tubules, and suppressed histological maturation in the epididymis and the prostate weight. These observations indicate that prenatally administered DES impairs testicular endocrine function continuously as well as putuitary function, but the induced low level of testosterone disrupts spermatogensis and permanently inhibits the morphological development of epididymis and prostate.
Ketoprofen has been reported to have such side effects as photosensitive dermatitis in humans (The Ministry of Health, Labour and Welfare, 2001). In the present study, as part of a safety evaluation of Miltax®, an application drug containing ketoprofen, phototoxicity of Miltax® was examined in guinea pigs. In the present skin phototoxicity study, Miltax® was applied for 12 hr. Ultraviolet (UV) rays were irradiated to examine whether or not Miltax® elicited skin reaction. Two kinds of UV-A plus UV-B dual irradiation and UV-A single irradiation were used for the elicitation. With UV-A plus UV-B dual irradiation on the Miltax® application site, no skin reaction was observed at UV irradiation side in any animals, in contrast to the case of the positive control article, 8-methoxypsoralen (8-MOP). Similar results were obtained with UV-A single irradiation. From these results, Miltax® that contained ketoprofen did not show any skin phototoxicity in the guinea pig.
Newborn rat studies were conducted with oral administration of 1,3-dibromopropane (DBP) and 1,1,2,2-tetrabromoethane (TBE) from postnatal Days 4 to 21 to allow comparison of NOAELs and unequivocally toxic levels with those from 28-day young rat studies starting at 5-6 weeks of age. The unequivocally toxic level was estimated by our specified criteria, requiring simultaneous change of organ weights, histopathology, some biochemical parameters and body weights, because in this study only hypertrophy of hepatocytes was observed as a major histopathological change. DBP caused centrilobular hypertrophy of hepatocytes with alteration in biochemical parameters, as well as lowering of body weights, regardless of sex, in both newborn and young rats. NOAELs and unequivocally toxic levels were considered to be 50 and 150 mg/kg/day in newborn rats and 10 and 250 mg/kg/day in young rats, respectively. In the newborn rat study of TBE, some hepatic effects observed at the top dose of 50 mg/kg were not considered adverse because of the lack of histopathological changes. Significant lowering of body weight was noted at 200 mg/kg in the dose-finding study but histopathological data were not available. In the young rat study, there was no definite toxicity at 6 mg/kg and hypertrophic changes in liver and thyroids without body weight change occurred at 200 mg/kg. There were no clear sex differences in both the newborn and young rat studies. NOAELs were considered to be 50 and 6 mg/kg/day in newborn and young rats, respectively, but unequivocally toxic levels for both rats could not be estimated. Abnormalities of external and sexual development and reflex ontogeny in the newborn were not observed with either chemical. Based on these results, it can be concluded that the target organ of DBP and TBE is the liver in both newborn and young rats, and that while the doses at which toxic signs began to appear are higher in newborn rats, those causing clear toxicity may be paradoxically lower in the newborn case.
- Anatomical evidence indicates that cholinergic and opioidergic systems are co-localized and acting on the same neuron. However, the regulatory mechanisms between cholinergic and opioidergic system have not been well characterized. In the present study, the potential involvement of μ-opioid receptors in mediating the changes of toxic signs and muscarinic receptor binding after administration of irreversible anti-acetylcholinesterase diisopropylfluorophosphate (DFP) was investigated. DFP (1 mg/kg/day, subcutaneous injection, s.c.)-induced tremors and chewing movements were monitored during the 28-day treatment period in μ-opioid receptor knockout and wild type mice. Autoradiographic studies of total, M1, and M2 muscarinic receptors were conducted using [3H]-quinuclidinyl benzilate, [3H]-pirenzepine, and [3H]-AF-DX384 as ligands, respectively. DFP-induced tremors in both μ-opioid receptor knockout and wild type mice showed tolerance development. However, DFP-induced tremors in μ-opioid receptor knockout mice showed delayed tolerance development than that of DFP-treated wild type controls. DFP-induced chewing movements in both μ-opioid receptor knockout and wild type mice failed to show development of tolerance after four weeks of treatment. M2 muscarinic receptor binding of DFP-treated μ-opioid receptor knockout mice was significantly decreased than that of the DFP-treated wild type controls in the striatum, but not in the cortex and hippocampus. However, there were no significant differences in total and M1 muscarinic receptor binding between DFP-treated μ-opioid receptor knockout and wild type mice in the cortex, striatum and hippocampus. These studies indicate that μ-opioid receptors play an important role through the striatal M2 muscarinic receptors to regulate the development of tolerance to DFP-induced tremors.
High throughput toxicological estimation is required for safety evaluation in the early stage of drug discovery. In this context, establishment of an in vitro screening system reflecting in vivo toxicity is demanded for earlier safety assessment. We investigated LDH release and mitochondrial respiration (WST-1 reduction assay; WST-1) to detect cytotoxicity, morphological evaluation, and proteomics for estimating the reliable and sensitive biomarkers by using rat primary hepatocytes exposed to the compounds (acetaminophen, amiodarone, tetracycline and carbon tetrachloride) that are known to induce hepatotoxicity. In LDH release, no significant difference was detected between the control and compound exposed cells after exposure for 3 or 6 hr, but a dose-dependent increase was observed after exposure for 24 hr. Regarding the WST-1 assay, a dose-dependent reduction was detected after exposure for 6 and 24 hr to all of the compounds evaluated. In the proteomics analysis, 31 candidate proteins were identified from among the 103 demonstrating altered expression spots after exposure to acetaminophen. It was concluded that the cytotoxicity was detected earlier by measuring WST-1 than by measuring LDH release because the reduction of mitochondrial respiration is an expressions of earlier toxicity for cellular function, while the measured increase in the LDH release occurs after the failure of the cell membrane. Mitochondrial respiration ability was a useful parameter for cytotoxicity in in vitro hepato-toxicity screening, as cytotoxicity can be detected during the early stage of exposure. In addition to the conventional biomarkers, several protein biomarkers which relate to oxidative stress and metabolism-regulation were detected. Further comprehensive analysis of defined proteins would be necessary to estimate the more sensitive toxicology biomarker.