Hepatic and renal toxicities and metabolism of fentanyl analogues in rats.

New synthetic opioids continue to emerge in the illicit market, and among them, fentanyl analogues pose a serious threat to the public health with their abuse and trafficking. We investigated the toxicity of fentanyl analogues on the liver and kidneys mediated by the µ-opioid receptor (MOR). Our study focused on 4-fluoro-isobutyrylfentanyl (4F-iBF), which is classified as a "narcotic" in Japan; structurally similar analogues 4-chloro-isobutyrylfentanyl (4Cl-iBF) and isobutyrylfentanyl (iBF) were also investigated. Rats that were intraperitoneally administered 4F-iBF (5 mg/kg (12.3 µmol/kg)) or iBF (12.3 µmol/kg) displayed hepatic and renal ischemic-like damage, but 4Cl-iBF (12.3 µmol/kg) did milder renal damage only. We found that the agonist activity of 4F-iBF, at MORs was approximately 7.2 times that of 4Cl-iBF, and that pretreatment with MOR antagonist naltrexone (0.8 mg/kg) alleviated liver and kidney injuries caused by 4F-iBF. These results suggested that 4F-iBF might cause ischemic damage to the liver and kidneys, induced by respiratory depression mediated by MORs. Furthermore, to elucidate the metabolism of fentanyl analogues, we investigated the change over time in the amount of 4F-iBF, 4Cl-iBF, iBF (6.15 µmol/kg, respectively), and their respective metabolites in serum after intraperitoneal administration to rats. The results showed that in 24-h post-dose serum, 4Cl-iBF and iBF were substantially eliminated while 4F-iBF remained at about 30% of the maximum level, and each of the N-dephenylethylated metabolites of 4F-iBF, 4Cl-iBF, and iBF was detected in 2-h post-dose serum. The results from this study revealed information on the hepatic and renal toxicities and metabolism related to fentanyl analogues.

Reproductive and neurobehavioral effects of maternal exposure to dinotefuran in the F1 -generation mice.

BACKGROUND: Few studies were found on the neurobehavioral toxicity of dinotefuran in mammals. This study was designed to evaluate the reproductive and neurobehavioral effects of maternal exposure to dinotefuran in mice. METHODS: Dinotefuran was given in the diet at levels of 0% (control), 0.012% (18.2-50.6 mg/kg body weight [bw]/day), 0.03% (44.2-125.8 mg/kg bw/day), and 0.075% (111.3-311.0 mg/kg bw/day) in females (10/group) during the gestation (gestation day 1-5 to 15-19) and lactation periods (lactation day 0 to 24-28). Selected reproductive and neurobehavioral parameters were measured in the F1 generation. RESULTS: Litter size and litter weight significantly increased in the high-dose group. The average body weight of male and female offspring increased in treatment groups at postnatal day (PND) 21. In female offspring, surface righting on PND 4 was delayed in higher-dose groups. Cliff avoidance on PND 7 was delayed in a dose-related trend. The route of olfactory orientation on PND 14 was delayed in the high-dose group. For exploratory behavior of the F1 generation, the frequencies of mice with urination increased in a dose-related trend in male offspring. In female offspring, the related variables of rearing increased in dose-related manners. The average rearing time lengthened and the number of defecations increased in the high-dose group in adult males. In adult females, the rearing time shortened in the high-dose group. Several variables differed in the high-dose group for spontaneous behavior in adult females. CONCLUSIONS: The dose levels of dinotefuran produced several adverse effects on neurobehavioral parameters in mice.

Two-year intermittent exposure of a multiwalled carbon nanotube by intratracheal instillation induces lung tumors and pleural mesotheliomas in F344 rats.

BACKGROUND: A mounting number of studies have been documenting the carcinogenic potential of multiwalled carbon nanotubes (MWCNTs); however, only a few studies have evaluated the pulmonary carcinogenicity of MWCNTs in vivo. A 2-year inhalation study demonstrated that MWNT-7, a widely used MWCNT, was a pulmonary carcinogen in rats. In another 2-year study, rats administered MWNT-7 by intratracheal instillation at the beginning of the experimental period developed pleural mesotheliomas but not lung tumors. To obtain data more comparable with rats exposed to MWNT-7 by inhalation, we administered MWNT-7 to F344 rats by intratracheal instillation once every 4-weeks over the course of 2 years at 0, 0.125, and 0.5 mg/kg body weight, allowing lung burdens of MWNT-7 to increase over the entire experimental period, similar to the inhalation study. RESULTS: Absolute and relative lung weights were significantly elevated in both MWNT-7-treated groups. Dose- and time-dependent toxic effects in the lung and pleura, such as inflammatory, fibrotic, and hyperplastic lesions, were found in both treated groups. The incidences of lung carcinomas, lung adenomas, and pleural mesotheliomas were significantly increased in the high-dose group compared with the control group. The pleural mesotheliomas developed mainly at the mediastinum. No MWNT-7-related neoplastic lesions were noted in the other organs. Cytological and biochemical parameters of the bronchoalveolar lavage fluid (BALF) were elevated in both treated groups. The lung burden of MWNT-7 was dose- and time-dependent, and at the terminal necropsy, the average value was 0.9 and 3.6 mg/lung in the low-dose and high-dose groups, respectively. The number of fibers in the pleural cavity was also dose- and time-dependent. CONCLUSIONS: Repeated administration of MWNT-7 by intratracheal instillation over the 2 years indicates that MWNT-7 is carcinogenic to both the lung and pleura of rats, which differs from the results of the 2 carcinogenicity tests by inhalation or intratracheal instillation.

Iron oxide nanoparticles exert inhibitory effects on N-Bis(2-hydroxypropyl)nitrosamine (DHPN)-induced lung tumorigenesis in rats.

Iron oxide nanoparticles (magnetite) have been widely used in industry and medicine. However, the safety assessment of magnetite has not been fully completed. The present study was conducted to assess effects of magnetite on carcinogenic activity, using a medium-term bioassay protocol. A total of 100 male Fischer 344 rats, 6 weeks old, were randomly divided into 5 groups of 20 animals each, and given a basal diet and drinking water containing 0 or 0.1% of N-bis(2-hydroxypropyl)nitrosamine (DHPN) for 2 weeks. Two weeks later, the rats were intratracheally instilled magnetite 7 times at an interval of 4 weeks, at the doses of 0, 1.0 or 5.0 mg/kg body weight, and sacrificed at the end of the experimental period of 30 weeks. The multiplicities of macroscopic lung nodules and histopathologically diagnosed bronchiolo-alveolar hyperplasia, induced by DHPN, were both significantly decreased by the high dose of magnetite. The expression of minichromosome maintenance (MCM) protein 7 in non-tumoral alveolar epithelial cells, and the number of CD163-positive macrophages in tumor nodules were both significantly reduced by magnetite. It is suggested that magnetite exerts inhibitory effects against DHPN-induced lung tumorigenesis, by the reduction of alveolar epithelial proliferation and the M2 polarization of tumor-associated macrophages.

A case of spontaneous Zymbal's gland carcinoma with lung metastasis in an aged Fischer 344 rat.

Zymbal's gland neoplasms are induced in rats through the administration of various carcinogens, but spontaneous neoplasia is rare. This report describes a spontaneous Zymbal's gland carcinoma with lung metastasis found in an aged male Fischer 344 rat. Macroscopically, the dome-like tumor nodule, approximately 30 mm in diameter with ulceration, was located near the ear canal of the rat. No healthy tissue or structure of Zymbal's gland was identified on the corresponding side, while the normal salivary glands and a lacrimal gland were observed. Histologically, a large part of the tumor mass was occupied by poorly differentiated neoplastic cells, the shapes of which were oval to polygonal or fusiform. Additionally, clusters of sebaceous-like foamy cells and squamous metaplasia with prominent keratinization were observed. Tumor cells were found to metastasize to the lung; these cells displayed histological similarities, including a sebaceous gland-like pattern, to those in the primary site. The tumor cells were immunohistochemically positive for cytokeratin AE1/AE3 or vimentin but negative for CD68, S100, α-smooth muscle actin, von Willebrand factor, and desmin. Our results indicate that the tumor was a poorly differentiated Zymbal's gland carcinoma with lung metastasis.

A histopathological analysis of spontaneous neoplastic and non-neoplastic lesions in aged male RccHan:WIST rats.

Histopathological information about spontaneous lesions in aged Hannover Wistar rats is limited. In this study, we describe spontaneous lesions found in 39 male RccHan:WIST rats used as a control in a carcinogenicity study. Neoplastic lesions were frequently seen in the endocrine system, such as pituitary adenomas in the pars distalis. This strain exhibited a high incidence of thymoma (10.3%), compared to other strains. We encountered an oligodendroglioma, a pituitary adenoma of the pars intermedia, and a prostate adenocarcinoma, which are comparatively rare in rats. While the variety and incidence of non-neoplastic lesions were similar to those in other strains, several interesting lesions occurred with relatively high incidence, including "harderianization" of the extraorbital lacrimal gland, common bile duct ectasia, and hyperplasia of pulmonary endocrine cells in the lung. Furthermore, comparative analyses demonstrated that the severity of chronic progressive nephropathy and murine progressive cardiomyopathy in RccHan:WIST rats was less than that in F344 rats.

Inhalation Exposure Method for Illegal Drugs.

We developed a new inhalation exposure method to evaluate effects of synthetic cannabimimetics that are being distributed as new, unregulated drugs in the Tokyo area. We selected the commercial product "SOUTOU" containing AB-CHMINACA and 5F-AMB as the test drug and dried marshmallow (Althaea officinalis) leaves as the negative control. A half cigarette packed with dried marshmallow leaves or SOUTOU was ignited, then mainstream smoke from each was delivered to five mice in an exposure box. After the cigarettes were fully consumed, neurobehavioral observations and a catalepsy test were performed at 15, 30 and 60 min after exposure. The effluent air from the exposure box was poured into impingers containing acetonitrile (first impinger) and dimethyl sulfoxide (second impinger). The resulting solutions were analyzed to assess decomposition of the synthetic cannabimimetics. Mice exposed to SOUTOU smoke showed many excitement behaviors and some suppressive behaviors at 15, 30 and 60 min. These clearly included cannabimimetic specific pharmacological actions. Negative control mice also showed some suppressive behaviors at 15 min but these were attenuated at later times, nearly disappearing at 60 min. In addition, the behavioral effects observed in controls were less pronounced than those in SOUTOU exposed mice. The inhalation exposure method developed in our study would be effective for determining cannabinoid specific pharmacological effects of illegal drugs, as well as for assessing the presence of active compound(s) by comparing the test substance with a negative control.

Cytotoxic effects of psychotropic benzofuran derivatives, N-methyl-5-(2-aminopropyl)benzofuran and its N-demethylated derivative, on isolated rat hepatocytes.

The novel psychoactive compounds derived from amphetamine have been illegally abused as recreational drugs, some of which are known to be hepatotoxic in humans and experimental animals. The cytotoxic effects and mechanisms of 5-(2-aminopropyl)benzofuran (5-APB) and N-methyl-5-(2-aminopropyl)benzofuran (5-MAPB), both of which are benzofuran analogues of amphetamine, and 3,4-methylenedioxy-N-methamphetamine (MDMA) were studied in freshly isolated rat hepatocytes. 5-MAPB caused not only concentration-dependent (0-4.0 mm) and time-dependent (0-3 h) cell death accompanied by the depletion of cellular ATP and reduced glutathione and protein thiol levels, but also accumulation of oxidized glutathione. Of the other analogues examined at a concentration of 4 mm, 5-MAPB/5-APB-induced cytotoxicity with the production of reactive oxygen species and loss of mitochondrial membrane potential was greater than that induced by MDMA. In isolated rat liver mitochondria, the benzofurans resulted in a greater increase in the rate of state 4 oxygen consumption than did MDMA, with a decrease in the rate of state 3 oxygen consumption. Furthermore, the benzofurans caused more of a rapid mitochondrial swelling dependent on the mitochondrial permeability transition than MDMA. 5-MAPB at a weakly toxic level (1 mm) was metabolized slowly: levels of 5-MAPB and 5-APB were approximately 0.9 mm and 50 µm, respectively, after 3 h incubation. Taken collectively, these results indicate that mitochondria are target organelles for the benzofuran analogues and MDMA, which elicit cytotoxicity through mitochondrial failure, and the onset of cytotoxicity may depend on the initial and/or residual concentrations of 5-MAPB rather than on those of its metabolite 5-APB. Copyright © 2016 John Wiley & Sons, Ltd.

Magnetite Nanoparticles Induce Genotoxicity in the Lungs of Mice via Inflammatory Response.

Nanomaterials are useful for their characteristic properties and are commonly used in various fields. Nanosized-magnetite (MGT) is widely utilized in medicinal and industrial fields, whereas their toxicological properties are not well documented. A safety assessment is thus urgently required for MGT, and genotoxicity is one of the most serious concerns. In the present study, we examined genotoxic effects of MGT using mice and revealed that DNA damage analyzed by a comet assay in the lungs of imprinting control region (ICR) mice intratracheally instilled with a single dose of 0.05 or 0.2 mg/animal of MGT was approximately two- to three-fold higher than that of vehicle-control animals. Furthermore, in gpt delta transgenic mice, gpt mutant frequency (MF) in the lungs of the group exposed to four consecutive doses of 0.2 mg MGT was significantly higher than in the control group. Mutation spectrum analysis showed that base substitutions were predominantly induced by MGT, among which G:C to A:T transition and G:C to T:A transversion were the most significant. To clarify the mechanism of mutation caused by MGT, we analyzed the formation of DNA adducts in the lungs of mice exposed to MGT. DNA was extracted from lungs of mice 3, 24, 72 and 168 h after intratracheal instillation of 0.2 mg/body of MGT, and digested enzymatically. 8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and lipid peroxide-related DNA adducts were quantified by stable isotope dilution liquid chromatography-mass spectrometry (LC-MS/MS). Compared with vehicle control, these DNA adduct levels were significantly increased in the MGT-treated mice. In addition to oxidative stress- and inflammation related-DNA adduct formations, inflammatory cell infiltration and focal granulomatous formations were also observed in the lungs of MGT-treated mice. Based on these findings, it is suggested that inflammatory responses are probably involved in the genotoxicity induced by MGT in the lungs of mice.

Genotoxicity of multi-walled carbon nanotubes in both in vitro and in vivo assay systems.

The genotoxic effects of multi-walled carbon nanotubes (MWCNTs) were examined by using in vitro and in vivo assays. MWCNTs significantly induced micronuclei in A549 cells and enhanced the frequency of sister chromatid exchange (SCE) in CHO AA8 cells. When ICR mice were intratracheally instilled with a single dose (0.05 or 0.2 mg/animal) of MWCNTs, DNA damage of the lungs, analysed by comet assay, increased in a dose-dependent manner. Moreover, DNA oxidative damage, indicated by 8-oxo-7,8-dihydro-2'-deoxyguanosine and heptanone etheno-deoxyribonucleosides, occurred in the lungs of MWCNT-exposed mice. The gpt mutation frequencies significantly increased in the lungs of MWCNT-treated gpt delta transgenic mice. Transversions were predominant, and G:C to C:G was clearly increased by MWCNTs. Moreover, many regions immunohistochemically stained for inducible NO synthase and nitrotyrosine were observed in the lungs of MWCNT-exposed mice. Overall, MWCNTs were shown to be genotoxic both in in vitro and in vivo tests; the mechanisms probably involve oxidative stress and inflammatory responses.

Long-term Pulmonary Responses to Quadweekly Intermittent Intratracheal Spray Instillations of Magnetite (Fe3O4) Nanoparticles for 52 Weeks in Fischer 344 Rats.

Information about potential risks of iron nanomaterials is still limited, while a wide variety of applications are expected. We recently reported acute phase responses of male and female Fischer 344 rats after a single intratracheal spray instillation of Fe3O4 nanoparticles (magnetite), clearly showing dose-dependent pulmonary inflammatory changes (Tada et al., J Toxicol Pathol 25, 233-239, 2012). The present study assessed long-term responses of male and female Fischer 344 rats to multiple administrations of magnetite. Ten-week-old male and female Fischer 344 rats (n=20/group) were exposed to a total of 13 quadweekly intermittent intratracheal spray instillations of magnetite during the experimental period of 52 weeks, at doses of 0, 0.2 (low), 1.0 (medium) and 5.0 (high-dose) mg/kg body weight per administration. Absolute and relative lung weights of the high-dose group were significantly higher than those of the control group. Macroscopically, slight enlargement and scattered black patches were recognized in the lungs and the lung-associated lymph nodes of the high-dose group. Histopathologically, infiltration of macrophages phagocytosing magnetite (all dose groups) and of chronic inflammatory cells (medium- and high-dose males and high-dose females), alveolar bronchiolization and granuloma (high-dose group) were observed. In addition, alveolar hyperplasias were observed in some rats of the high-dose group, and cytoplasmic overexpression of ß-catenin protein was immunohistochemically found in such lesions. The present results clearly show that instilled magnetite causes chronic inflammatory responses in the lung. These responses occur in a dose-dependent manner without apparent differences among sexes.

Acute phase pulmonary responses to a single intratracheal spray instillation of magnetite (fe(3)o(4)) nanoparticles in Fischer 344 rats.

Iron nanomaterials are of considerable interest for application to nanotechnology-related fields including environmental catalysis, biomedical imaging, drug delivery and hyperthermia, because of their superparamagnetic characteristics and high catalytic abilities. However, information about potential risks of iron nanomaterials is limited. The present study assessed pulmonary responses to a single intratracheal spray instillation of triiron tetraoxide nanoparticles (magnetite) in rats. Ten-week-old male and female Fischer 344 rats (n=5/group) were exposed to a single intratracheal spray instillation of 0 (vehicle), 5.0, 15.0 or 45.0 mg/kg body weight (BW) of magnetite. After 14 days, the rats were sacrificed, and biological consequences were investigated. The lung weights of the 15.0 and 45.0 mg/kg BW male and female groups were significantly higher than those of the control groups. The lungs of treated rats showed enlargement and black patches originating from the color of magnetite. The typical histopathological changes in the lungs of the treated rats included infiltration of macrophages phagocytosing magnetite, inflammatory cell infiltration, granuloma formation and an increase of goblet cells in the bronchial epithelium. The results clearly show that instilled magnetite causes foreign body inflammatory and granulating lesions in the lung. These pulmonary responses occur in a dose-dependent manner in association with the increase in lung weight.

A 90-day Feeding Toxicity Study of l-Serine in Male and Female Fischer 344 Rats.

A subchronic feeding study of l-serine (l-Ser) was conducted with groups of 10 male and 10 female Fischer 344 rats fed a powder diet containing 0, 0.06, 0.5, 1.5 or 5.0% concentrations of l-Ser for 90 days. There were no toxicologically significant, treatment-related changes with regards to body weight, food intake, water intake or urinalysis data. In several of the hematology, serum biochemistry and organ weight parameters, significant changes were observed between some of the treated groups and the controls. All these changes, however, were subtle and lacked any corresponding pathological findings. In addition, the increased or decreased values remained within the range of the historical control values. In fact, histopathological assessment revealed only sporadic and/or spontaneous lesions. In conclusion, the no-observed-adverse-effect-level (NOAEL) for l-Ser was, therefore, determined to be at least a dietary dose of 5.0% (2765.0 mg/kg body weight/day for males and 2905.1 mg/kg body weight/day for females) under the present experimental conditions.

[A 104-week feeding study of genetically modified soybeans in F344 rats].

A chronic feeding study to evaluate the safety of genetically modified glyphosate-tolerant soybeans (GM soybeans) was conducted using F344 DuCrj rats. The rats were fed diet containing GM soybeans or Non-GM soybeans at the concentration of 30% in basal diet. Non-GM soybeans were a closely related strain to the GM soybeans. These two diets were adjusted to an identical nutrient level. In this study, the influence of GM soybeans in rats was compared with that of the Non-GM soybeans, and furthermore, to assess the effect of soybeans themselves, the groups of rats fed GM and Non-GM soybeans were compared with a group fed commercial diet (CE-2). General conditions were observed daily and body weight and food consumption were recorded. At the termination (104 weeks), animals were subjected to hematology, serum biochemistry, and pathological examinations. There were several differences in animal growth, food intake, organ weights and histological findings between the rats fed the GM and/or Non-GM soybeans and the rats fed CE-2. However, body weight and food intake were similar for the rats fed the GM and Non-GM soybeans. Gross necropsy findings, hematological and serum biochemical parameters, and organ weights showed no meaningful difference between rats fed the GM and Non-GM soybeans. In pathological observation, there was neither an increase in incidence nor any specific type of nonneoplastic or neoplastic lesions in the GM soybeans group in each sex. These results indicate that long-term intake of GM soybeans at the level of 30% in diet has no apparent adverse effect in rats.

[A 52-week feeding study of genetically modified soybeans in F344 rats].

A chronic feeding study to evaluate the safety of the genetically modified glyphosate-tolerant soybeans (GM soybeans) was conducted using rats. F344 DuCrj rats were fed diet containing GM soybeans or Non-GM soybeans at the concentration of 30% in basal diet. Non-GM soybeans were closely related strain of GM soybeans. These two diets were adjusted to an identical nutrient level. In this study, the influence of GM soybeans on rats was compared with that of the Non-GM soybeans, and furthermore, to assess the effect of soybeans themselves, the groups of rats fed GM and Non-GM soybeans were compared with a group fed commercial diet (CE-2). General conditions were observed daily and body weight and food consumption were recorded. At the intermediate examination (26 weeks), and at the termination (52 weeks), animals were subjected to hematology, serum biochemistry, and pathological examination. There were several differences in animal growth, food intake, serum biochemical parameters and histological findings between the rats fed the GM and/or Non-GM soybeans and the rats fed CE-2. However, body weight and food intake were similar for the rats fed the GM and Non-GM soybeans. Gross necropsy findings, hematological and serum biochemical parameters, organ weights, and pathological findings showed no meaningful difference between rats fed the GM and Non-GM soybeans. These results indicate that long-term intake of GM soybeans at the level of 30% in diet has no apparent adverse effect in rats.

Flame retardant tetrabromobisphenol A induced hepatic changes in ICR male mice.

Tetrabromobisphenol A (TBBPA) is widely used throughout the world as flame retardant for electronic equipment or building materials, and is detected in air at the dismantling plant, sewage sludge, sediment or human serum samples. In the present study, we examined the effects of TBBPA on the liver when administered to mice for 14 consecutive days. Groups of 7 (control group) or 8 (treated group) Crlj:CD1 (ICR) male mice were given 0 (control), 350, 700 or 1400mg/kg body weight/day TBBPA (99.1% pure) in olive oil for 14 days. The serum concentration of total-cholesterol in high-dose (1400mg/kg BW) group was higher than those of the control group. Absolute and relative liver weights were dose-dependently increased, and were significantly increased in high-dose (1400mg/kg BW) group. The histological findings showed that the slight enlargement of the hepatocytes, inflammatory cell infiltrations and focal necrosis of hepatocytes were more marked in liver of treated groups (from 350mg/kg BW) than in control group. The present data suggest the possibility of inducing hepatic lesion by TBBPA dosing.

Effects of N-nitrosofenfluramine, a component of Chinese dietary supplement for weight loss, on CD-1 mice.

Many cases of hepatopathy including deaths have frequently occurred after ingestion of Chinese dietary supplements for weight loss containing N-nitrosofenfluramine (N-fen), a nitroso derivative of fenfluramine (Fen), which was used for the treatment of obesity in the United States. Since Fen decreases appetite by decreasing the serotonin level and exhibits an antibiotic effect, N-fen may have been added, expecting a similar effect. Thus, we synthesized N-fen and orally administered it to mice, and investigated its effect on the liver as well as on the cerebral serotonin nervous system to investigate whether N-fen exhibits an anorectic effect. Three doses of N-fen were orally administered once daily to mice for 1 week. No significant changes in body weight, food intake, and general condition were noted. The liver and kidney weights were significantly increased. On blood chemistry, alkaline phosphatase, aspartate aminotransferase, and alanine aminotransferase activities were increased, and total bilirubin and albumin were slightly decreased. On histopathological examination, acidophilic changes and mild cellular swelling were noted in the liver. The liver drug-metabolizing enzyme (P-450) level was significantly higher. The effect of N-fen on the serotonin (5HT) nervous system was examined by quantitative autoradiography of the mouse brain, and it was found that N-fen did not decrease the 5HT nerve activity. Effects of reuptake and release of monoamine neurotransmitters [dopamine (DA), 5HT, and norepinephrine (NE)] were investigated. N-fen inhibited a little 5HT reuptake, and did not inhibit reuptakes of DA and NE. Moreover, N-fen did not affect release of the three monoamines. The above findings suggested that N-fen did not exhibit a serotonin nerve fiber-mediated anorectic effect in mice, but induced hepatopathy.